Films for controlled cell growth and adhesion

ABSTRACT

An article for controlling the attachment and growth of cells on a surface of the article, and a method for the use of the article is provided. The article comprises a substratum having a surface and a film on the surface, the film comprising a network of a net positively charged composition and a net negatively charged composition, wherein the net positively charged composition comprises a net positively charged polyelectrolyte or the net negatively charged composition comprises a net negatively charged polyelectrolyte, and the net positively charged polyelectrolyte or the net negatively charged polyelectrolyte contain (i) a polymer repeat unit having at least two fluorine atoms, or (ii) a polymer repeat unit having a zwitterion group. The method comprises contacting the article with living tissue, living organisms, or with water in an aqueous system comprising living organisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional application Ser.No. 60/571,818, filed on May 17, 2004, the contents of which areincorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant DMR 9727717awarded by the National Science Foundation. The Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

This invention relates generally to the field of cell growth and, moreparticularly, to using polyelectrolyte complex films to coat surfaces toenhance or inhibit cell growth, adhesion, and differentiation.

In order for cells to adhere and grow on a substrate, the interfacebetween the substrate and the cell growth medium must possess anappropriate combination of physical and chemical properties. Controlover the surface of substrate provides for control over cell adhesion.Advantageous properties imparted by a surface range from the completerejection of any cell adhesion or growth, to cell adhesion withoutgrowth, to cell adhesion, growth, and differentiation. The propertydesired depends on the end-use of the substrate. For example, articlesimplanted in vivo, such as stents, catheters, and artificial organs,preferably do not induce biochemical processes that lead to scarringand/or rejection of said article. These implants may be advantageouslycoated with thin films that render them biocompatible. Alternatively,some applications, especially those in tissue engineering, requiresubstrates that encourage the growth, differentiation, and proliferationof cells. A strategy for modifying the cell adhesion and cell growthproperties of surfaces is needed.

Polyelectrolytes are macromolecules comprising a plurality of chargedrepeat units. Amorphous complexes may be formed by contacting solutionsof polyelectrolytes bearing opposite charges. The driving force forassociation, or complexation, of polyelectrolytes is multiple ionpairing between oppositely charged repeat units on different molecules.

Recently, thin films of polyelectrolyte complexes have been preparedusing polyelectrolytes which are alternately deposited on a substrate orsubstratum. See Decher and Schlenoff, Eds., Multilayer ThinFilms—Sequential Assembly of Nanocomposite Materials, Wiley-VCH,Weinheim (2003); Decher, Science, 277, 1232 (1997). Decher and Hong(U.S. Pat. No. 5,208,111) disclose a method for a buildup of multilayersby alternating dipping, i.e., cycling a substrate between two reservoirscontaining aqueous solutions of polyelectrolytes of opposite charge,with an optional rinse step in polymer-free solution following eachimmersion. Each cycle adds a layer of polymer via ion pairing forces tothe oppositely-charged surface and reverses the surface charge therebypriming the film for the addition of the next layer. Films prepared inthis manner tend to be uniform, follow the contours and irregularitiesof the substrate, and are typically between about 10 nm and about 10,000nm thick. The thickness of a film depends on many factors, including thenumber of layers deposited, the ionic strength of the solutions, thetypes of polymers, the deposition time, the solution pH, thetemperature, and the solvent used. Although studies have shown thatsubstantial interpenetration of the individual polymer layers results inlittle composition variation over the thickness of a film, such polymerthin films are, nevertheless, referred to as polyelectrolyte multilayers(PEMUs).

Surface modification using polyelectrolyte multilayers to developbiocompatible materials has been attracting attention lately due to theease of synthesis and cost-effectiveness of the layer-by-layertechnique. See Decher, G., Schlenoff, J. B. Multilayer ThinFilms—Sequential Assembly of Nanocomposite Materials; Wiley-VCH:Weinheim, Germany, 2003. Surface properties ranging from hydrophobic tohydrophilic, charged to uncharged, and smooth to rough can be generatedusing a variety of parameters including the chemical nature of thepolyelectrolytes and the pH, ionic strength, and temperature used formultilayer synthesis. Because proteins play an important role in theadhesion, spreading, and growth of cells, considerable effort has beenexpended in developing polyelectrolyte thin films with properties thatmake the surface adhesive or resistant to protein adsorption. SeeMuller, M.; Rieser, T.; Kothe, M.; Kessler, B.; Brissova, M.; Lunkwitz,K. Macromol. Symp. 1999, 145, 149, Muller, M.; Brissova, M.; Rieser, T.;Powers, A. C.; Lunkwitz, K. Mat. Sci. Eng. C-Bio. S. 1999, 8-9, 163,Muller, M.; Rieser, T.; Lunkwitz, K.; Meier-Haack, J. Macromol. Rapid.Comm. 1999, 20, 607, Ladam, G.; Gergely, C.; Senger, B.; Decher, G.;Voegel, J. C.; Schaaf, P.; Cuisinier, F. J. G. Biomacromolecules 2000,1, 674, Ladam, G.; Schaaf, P.; Cuisinier, F. J. G.; Decher, G.; Voegel,J. C. Langmuir 2001, 17, 878, and Salloum, D. S.; Schlenoff, J. B.Biomacromolecules 2004. Although an understanding of PEMU-proteinadsorption is necessary to intelligently engineer cell-biomaterialinteraction, it is difficult to predict PEMU-cell biocompatibility fromsimple measurements of protein adsorption. See Han, D. K.; Ryu, G. H.;Park, K. D.; Jeong, S. Y.; Kim, Y. H.; Min, B. G. Journal ofBiomaterials Science-Polymer Edition 1993, 4, 401, Ostuni, E.; Chapman,R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides,G. M. Langmuir 2001, 17, 6336, and Mendelsohn, J. D.; Yang, S. Y.;Hiller, J.; Hochbaum, A. I.; Rubner, M. F. Biomacromolecules 2003, 4,96. Recent investigations using cultured cells revealed PEMU propertiesimportant for cell biocompatibility. See Ito, Y.; Chen, G. P.; Imanishi,Y. Bioconjugate Chemistry 1998, 9, 277, Chluba, J.; Voegel, J. C.;Decher, G.; Erbacher, P.; Schaaf, P.; Ogier, J. Biomacromolecules 2001,2, 800, Tryoen-Toth, P.; Vautier, D.; Haikel, Y.; Voegel, J. C.; Schaaf,P.; Chluba, J.; Ogier, J. Journal of Biomedical Materials Research 2002,60, 657, Richert, L.; Lavalle, P.; Vautier, D.; Senger, B.; Stoltz, J.F.; Schaaf, P.; Voegel, J. C.; Picart, C. Biomacromolecules 2002, 3,1170, Boura, C.; Menu, P.; Payan, E.; Picart, C.; Voegel, J. C.; Muller,S.; Stoltz, J. F. Biomaterials 2003, 24, 3521, Elbert, D. L.; Herbert,C. B.; Hubbell, J. A. Langmuir 1999, 15, 5355, and Serizawa, T.;Yamaguchi, M.; Akashi, M. Biomacromolecules 2002, 3, 724. Theseinvestigations have demonstrated that surfaces can be renderedcytophilic or cytophobic by embedding or attaching protein or peptidesto the multilayer and by tuning the pH used for multilayer buildup. SeeJessel, N.; Atalar, F.; Lavalle, P.; Mutterer, J.; Decher, G.; Schaaf,P.; Voegel, J. C.; Ogier, J. Adv. Mater. 2003, 15, 692 and Berg, M. C.;Yang, S. Y.; Hammond, P. T.; Rubner, M. F. Langmuir 2004, 20, 1362.Other modifications such as chemical cross-linking have improved thePEMU stability and cell adhesion. See Richert, L.; Boulmedais, F.;Lavalle, P.; Mutterer, J.; Ferreux, E.; Decher, G.; Schaaf, P.; Voegel,J. C.; Picart, C. Biomacromolecules 2003. Certain surfaces such aspolysaccharide films made by layer-by-layer buildup have beeninvestigated for use as antimicrobial coatings and bioactiveendovascular stent coatings. See Richert, L.; Lavalle, P.; Payan, E.;Shu, X. Z.; Prestwich, G. D.; Stoltz, J. F.; Schaaf, P.; Voegel, J. C.;Picart, C. Langmuir 2004, 20, 448 and Thierry, B.; Winnik, F. M.; Merhi,Y.; Silver, J.; Tabrizian, M. Biomacromolecules 2003, 4, 1564. In fact,polyelectrolyte complexes have a long history of use in preparingbioinert surfaces. See Tsuchida, E.; Abe, K. Advances in Polymer Science1982, 45, 1, Philippe, B.; Dautzenberg, H.; Linow, K. J.; Kotz, J.;Dawydoff, W. Progress in Polymer Science 1989, 14, 91, and Petrak, K.Journal of Bioactive and Compatible Polymers 1993, 8, 178.

The surface properties of endovascular stents may play an important rolein the process of restenosis. During restenosis, vascular smooth musclecells migrate to cover implanted stents, often building layers of tissuethat cause occlusion of the blood flow. See Indolfi, C.; Mongiardo, A.;Curcio, A.; Torella, D. Trends in Cardiovascular Medicine 2003, 13, 142.In evaluating biocompatibility, therefore, it is important to understandhow smooth muscle cells interact with PEMU surfaces. Smooth muscle cellsare capable of alternating between a ‘contractile’ phenotype,characterized by a non-motile cell type that possesses both acontractile smooth muscle cytoskeleton and a non muscle cytoskeleton forcell support, and a ‘synthetic’ phenotype that is motile and possesses anon-muscle cytoskeleton used for cell support and cell motility. SeeWorth, N. F.; Rolfe, B. E.; Song, J.; Campbell, G. R. Cell Motility andthe Cytoskeleton 2001, 49, 130 and Halayko, A. J.; Solway, J. Journal ofApplied Physiology 2001, 90, 358. The two cell phenotypes can be readilydistinguished by the cell shape, stability of adhesion, and organizationof the underlying cytoskeleton structures.

SUMMARY OF THE INVENTION

Among the aspects of this invention may be noted the provision ofarticles having thin films of polyelectrolyte complex thereon andmethods for use of said articles. The thin films compriseinterpenetrating networks of net positively charged compositions and netnegatively charged compositions, the compositions comprisingpolyelectrolytes containing polymer repeat units having at least twofluorine atoms, polymer repeat units having zwitterions groups, or both.Said articles having thin films thereon are adapted to promote celladhesion and growth, or to inhibit cell adhesion in living tissue or inmarine environments.

Briefly, therefore, the invention is directed to an article adapted foruse in combination with living tissue or in a marine environment, thearticle comprising: a substratum having a surface; and a polyelectrolytefilm on the surface, the polyelectrolyte film comprising a network of anet positively charged composition and a net negatively chargedcomposition, wherein the net positively charged composition comprises anet positively charged polyelectrolyte or the net negatively chargedcomposition comprises a net negatively charged polyelectrolyte, and thenet positively charged polyelectrolyte or the net negatively chargedpolyelectrolyte contain (i) a polymer repeat unit having at least twofluorine atoms, or (ii) a polymer repeat unit having a zwitterion group;wherein the substratum has a composition and shape adapting the articlefor use in combination with living tissue or in a marine environment.

The invention is further directed to an article adapted for use incombination with living tissue or organisms, the article comprising apolyelectrolyte film, the film comprising an interpenetrating network ofa net positively charged polyelectrolyte and a net negatively chargedpolyelectrolyte, the film having first and second surface regions with anet positively charged or net negatively charged polyelectrolyte exposedat each of said first and second surface regions wherein (1) the netpositively charged or negatively charged polyelectrolyte exposed in saidfirst surface region contains a polymer repeat unit having at least twofluorine atoms and (2) the net positively charged or negatively chargedpolyelectrolyte exposed in said second surface region contains a polymerrepeat unit having a zwitterion group.

The invention is yet further directed to a method for controlling theattachment and growth of cells on a surface of an article, the methodcomprising contacting the article with living tissue, living organisms,or with water in an aqueous system comprising living organisms whereinthe article comprises a substratum having a surface and a film on thesurface, the film comprising a network of a net positively chargedcomposition and a net negatively charged composition, wherein the netpositively charged composition comprises a net positively chargedpolyelectrolyte or the net negatively charged composition comprises anet negatively charged polyelectrolyte, and the net positively chargedpolyelectrolyte or the net negatively charged polyelectrolyte contain(i) a polymer repeat unit having at least two fluorine atoms, or (ii) apolymer repeat unit having a zwitterion group.

Other objects and aspects of the invention will be, in part, pointed outand, in part, apparent hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an ATR-FTIR spectrum showing the buildup of layers accordingto the method of Example 3. A corresponds to PDADMA, B corresponds to(PDADMA/P2PSVP), C corresponds to (PDADMA/P2PSVP/PAA), D corresponds to(PDADMA/P2PSVP/PAA/P2PSVP), E corresponds to(PDADMA/P2PSVP/PAA/P2PSVP/PDADMA). The negative peak in E indicates lossof the zwitterion P2PSVP.

FIG. 2 is an ATR-FTIR spectrum showing the buildup of PAH/PAA-co-PAEDAPSPEMU according to the method of Example 4. The graph shows thatlayer-by-layer buildup is achievable using a copolymer, which was notthe case when using the pure zwitterion polymer.

FIG. 3 is a graph of ellipsometric data showing thickness vs. number oflayers for the buildup of poly(acrylicacid)-co-poly(3-[2-(acrylamido)-ethyldimethyl ammonio]propanesulfonate), PAA-co-PAEDAPS, and PAH according to the method of Example4.

FIGS. 4A-4F are Axiocam phase images showing live A7r5 cells cultured on(A)(PFPVP/Nafion)₂, (B) (PDADMA/PSS)₂, (C) (PAH/PAA)₂, (D)(PFPVP/Nafion)₂PFPVP, (E) (PDADMA/PSS)₂PDADMA, and (F) (PAH/PAA)₂PAHaccording to the method of Example 6. Top panel are negatively chargedsurfaces (A-C). Bottom panel are positively charged surfaces (D-F).Hydrophobicity decreases from the left (A, D) panel to the right panel(C, F). Right bottom tags represent polymer on outermost surface (scalebar=20 μm).

FIGS. 5A-5B are Axiocam phase images showing live A7r5 cells cultured ondiblock polymers of (A)(PM2VP-b-PEO/PMA-b-PEO)₂PM2VP-b-PEO and (B)(PM2VP-b-PEO/PMA-b-PEO)₂ according to the method of Example 7. Rightbottom tags represent outermost surface (scale bar=20 μm).

FIGS. 6A-6C are Axiocam phase images showing live A7r5 cells cultured on(A)(PAH/PAA)₂ (B) a 90:10 mol % copolymer mixture of PAA:AEDAPS, and (C)a 75:25 mol % copolymer mixture of PAA:AEDAPS according to the method ofExample 8. Right bottom tags represent outermost surface (scale bar=20μm).

FIGS. 7A-7C are Axiocam phase images showing micropatterning of A7r5cells grown on 20 μm wide ridges of Nation® stamped on 80 μm widetroughs of 75:25 mol % PAA:PAEDAPS copolymer according to the method ofExample 9. (A) Fluorescently labeled phalloidin staining actin, (B) cellnuclei fluorescently labeled with DAPI, and (C) a phase image of thesame micropatterned area (scale bar=80 μm).

FIGS. 8A-8C are Axiocam phase images showing, according to the method ofExample 10, (A) A7r5 cells growing on Nation® (right) stamped ontoPAA:PAEDAPS (75:25 mol %) copolymer (left) showing the interface wherethe cell adhesive (Nation®) stamped surface meets the cell repulsivebackground surface (scale bar=20 μm), (B) A7r5 cells growing on Nation®(right) stamped onto (PAH/PAA)₂PAH (left) (scale bar=20 μm), and (C)Fluorescently labeled phalloidin staining actin in A7r5 cells growing atthe interface between the Nafion® stamp (right) and PAH background(left) (scale bar=10 μm).

FIGS. 9A-9E are Axiocam phase images showing unactivated human plateletsadhering to polyelectrolyte coated cover slips: (A) (PAH/PAA)₂PAH, (B)(PFPVP/Nafion)₂, (C) (PAH/PAA)₂ (D) a 90:10 mol % copolymer mixture ofPAA:AEDAPS, and (E) a 75:25 mol % copolymer mixture of PAA:AEDAPSaccording to the method of Example 11. Platelets are stained with Alexa488 phalloidin. (bar=2.5 μm).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

The articles of the present invention are uniquely adapted for use inliving tissue or aqueous systems comprising living organisms, saidarticles typically comprising a substratum and a polyelectrolyte film.The polyelectrolyte film comprises a network of a net positively chargedcomposition and a net negatively charged composition. The net positivelycharged composition may be, for example, a net positively chargedparticle or polyelectrolyte and the net negatively charged compositionmay be, for example, a net positively charged particle orpolyelectrolyte. In one preferred embodiment, the net positively chargedcomposition comprises a net positively charged polyelectrolyte and thenet negatively charged composition comprises a net negatively chargedpolyelectrolyte and the polyelectrolyte film is an interpenetratingnetwork of the two polyelectrolytes. The composition of thepolyelectrolyte may be varied to render the article suitable forenhancing the attachment and growth of cells in living tissue andaqueous systems, or conversely the composition of the polyelectrolytemay be varied to inhibit the attachment and growth of cells.Compositions of the present invention having surface regions comprisingpolyelectrolytes containing a polymer repeat unit having at least twofluorine atoms are uniquely suited to enhance cell attachment, growth,and even differentiation. Conversely, compositions having surfaceregions comprising polyelectrolytes containing a polymer repeat unithaving a zwitterion group are suited to inhibit cell adhesion. Thesubstratum has a composition and shape adapting the article for use incombination with living tissue or in a marine environment.

The articles comprising polyelectrolyte films disposed on a substrate oras free membranes which are designed to promote cell adhesion and growthmay comprise net negatively charged fluorinated compositions, netpositively charged fluorinated compositions, or both. Preferably, thepolyelectrolyte film comprises both net positively and net negativelycharged fluorinated compositions.

The net positively and net negatively charged fluorinated compositionsfor use in the polyelectrolyte films may comprise net positively and netnegatively charged polyelectrolytes comprising fluorinated chargedpolymer repeat units (e.g., fluorinated and positively charged groups orfluorinated and negatively charged groups), fluorinated uncharged orneutral polymer repeat units, unfluorinated charged polymer repeat units(e.g., positively charged groups or negatively charged groups), orunfluorinated uncharged or neutral polymer repeat units (e.g.,unfluorinated uncharged groups or unfluorinated zwitterion groups). Thevarious types of repeat units are present in random, block, or graftco-polymers, or as homopolymers. Fluorinated polymer repeat units,either charged or uncharged, preferably comprise fluorine groups presentas fluorinated conjugated groups, ═CF—, fluorinated methylene groups,—CF₂—, or fluorinated methyl groups, —CF₃. These moieties may be presentin fluorinated aliphatic groups, fluorinated olefinic groups,fluorinated aryl groups, or fluorinated heteroaryl groups. Thefluorinated polymer repeat unit comprises at least 2 fluorine atoms.Typically, the fluorinated polymer repeat unit comprises between 2 and21 fluorine atoms, more typically between 2 and 17 fluorine atoms, andeven more typically between 2 and 13 fluorine atoms. Preferably, atleast 5% of the polymer repeat units of the fluorinated polyelectrolytecomprise fluorinated groups. More preferably, at least 20% of thepolymer repeat units comprise fluorinated groups.

The polyelectrolyte films comprising fluorinated repeat units of thepresent invention comprise at least two or more alternating layers, eachlayer characterized by a charge. For example, thin films can be built upby depositing a first layer having a net charge, then depositing asecond layer having a net charge opposite that of the first layer. Atleast one of the layers comprises a net charged polyelectrolyte. Forexample, the thin film may comprise a blend of a net positively chargedpolyelectrolyte and a net negatively charged polyelectrolyte in aninterpenetrating network. In another example, the net positively chargedpolyelectrolyte may comprise one net positively charged polyelectrolyteor a blend of two or more net positively charged polyelectrolytes, atleast one positively charged polyelectrolyte containing fluorinatedrepeat units. Also, the net negatively charged polyelectrolyte maycomprise one negatively charged polyelectrolyte or a blend of two ormore net negatively charged polyelectrolytes, at least one negativelycharged polyelectrolyte containing fluorinated repeat units. The thinfilms of the present invention may comprise a blend of two or more netpositively charged and a blend of two or more net negatively chargedpolyelectrolytes. The blended polyelectrolytes preferably comprise atleast one net positively charged polyelectrolyte which containsfluorinated repeat units, at least one net negatively chargedpolyelectrolyte which contains fluorinated repeat units, or both.Preferably, in such blends, at least 5% of the charged polyelectrolytescomprise fluorinated repeat units, and more preferably at least 20% ofthe polyelectrolytes in the blend comprise fluorinated repeat units.

In another embodiment, the polyelectrolyte film comprises net positivelycharged and net negatively charged compositions comprising a fluorinatedparticle having a charge. The polyelectrolyte film also comprises afluorinated polyelectrolyte having an opposite charge of the fluorinatedparticle to form a composite of particles and polyelectrolyte. Forexample, particles may be formed of polytetrafluoroethylene (PTFE) oranother polymer comprising polymer repeat units having at least twofluorine atoms. Preferably, the surface of the particles is constitutedof polymers comprising polymer repeat units having at least two fluorineatoms, while the contents of the bulk of the particles are not narrowlycritical. For example, the bulk of the particles may comprisefluorinated polymers, non-fluorinated polymers, or may even be hollow.Particles having an average particle size of less than about 100 nm tendto form stable colloids in water whereas particles having an averageparticle size of greater than about 100 nm tend to form unstablesuspensions in water. These dispersions, whether colloidal or not, canbe further stabilized by coating the particles with charged surfactants,thus imparting a net charge on the particles. The surfactants may bepositively charged or negatively charged. Representative surfactantsinclude alkyl sulfonates, alkyl sulfates such as sodium dodecyl sulfate,tetraalkylammonium such as alkyltrimethylammonium, or cetyl pyridinium.Particles constituted of polymers comprising polymer repeat units havingat least two fluorine atoms at the surface and coated with a chargedsurfactant may be used as a charged layer in a thin film.

The articles comprising polyelectrolyte films disposed on a substrate oras free membranes which are designed to inhibit cell adhesion maycomprise net negatively charged compositions comprising zwitteriongroups, net positively charged compositions comprising zwitteriongroups, or both. Preferably, the polyelectrolyte film comprises both netpositively and net negatively charged zwitterionic compositions.

The net positively charged and net negatively charged compositions foruse in polyelectrolyte films which inhibit cell adhesion may comprisenet positively charged and net negatively charged polyelectrolytescomprising polymer repeat units having zwitterion groups, chargedpolymer repeat units, or neutral polymer repeat units. The various typesof polymer repeat units are present in random, block, or graftco-polymers. Preferably, the polyelectrolytes comprise both zwitterionrepeat units and charged repeat units. More preferably, thepolyelectrolytes comprise zwitterion repeat units and carboxylic acidrepeat units. In such co-polymers, the zwitterion repeat unitsconstitute at least about 15 mole % of the co-polymer, preferably about20 mole % to about 70 mole % of the co-polymer, more preferably betweenabout 30 mole % to about 50 mole %.

The polyelectrolyte films comprising polymer repeat units havingzwitterion groups comprise at least two or more alternating layers, eachlayer characterized by a charge. For example, thin films can be built upby depositing a first layer having a net charge, then depositing asecond layer having a net charge opposite that of the first layer. Atleast one of the layers comprises a net charged polyelectrolyte. Forexample, the thin film may comprise a blend of a net positively chargedpolyelectrolyte and a net negatively charged polyelectrolyte in aninterpenetrating network. In another example, the net positively chargedpolyelectrolyte may comprise one net positively charged polyelectrolyteor a blend of two or more net positively charged polyelectrolytes, atleast one positively charged polyelectrolyte containing zwitterionrepeat units. Also, the net negatively charged polyelectrolyte maycomprise one negatively charged polyelectrolyte or a blend of two ormore net negatively charged polyelectrolytes, at least one negativelycharged polyelectrolyte containing zwitterion repeat units. The thinfilms may comprise a blend of two or more net positively charged and ablend of two or more net negatively charged polyelectrolytes. Theblended polyelectrolytes preferably comprise at least one net positivelycharged polyelectrolyte which contains zwitterion repeat units, at leastone net negatively charged polyelectrolyte which contains zwitterionrepeat units, or both.

In another embodiment, the thin film comprises additional agents whichfurther inhibit the adhesion of cells to the thin film comprisingzwitterions repeat units. Such agents include paclitaxel and sirolimus.The articles adapted for use in combination with living tissue ororganisms are characterized by a polyelectrolyte film having particularsurface characteristics. In general, the article comprises apolyelectrolyte film comprising a network of a net positively chargedcomposition and a net negatively charged composition. The net positivelycharged composition or the net negatively charged composition maycomprise charged particles provided, however, the net negatively chargedcomposition comprises a net negatively charged polyelectrolyte or thenet positively charged composition comprises a net positively chargedpolyelectrolyte. In one preferred embodiment, the film is characterizedby surface regions in which the net positively charged or net negativelycharged polyelectrolyte exposed at the surface regions contains apolymer repeat unit having at least two fluorine atoms or a polymerrepeat unit having a zwitterion group. It has been discovered that filmmodification such that the surface regions comprising the polymer repeatunit having at least two fluorine atoms or the polymer repeat unithaving a zwitterion group is sufficient to control the attachment andgrowth of cells and that the characteristics of the bulk of the film arenot narrowly critical to the film's cell attachment properties. Thepolyelectrolyte films may be present on substratum having a compositionand shape adapting the article for use in combination with living tissueor in a marine environment, or the films may be present as freemembranes. In one embodiment, the free membrane is a membrane havingopposing sides, the first surface region being one of the opposing sidesand the second surface region being the other opposing side. The netpositively charged or negatively charged polyelectrolyte exposed in saidfirst surface region contains a polymer repeat unit having at least twofluorine atoms and the net positively charged or negatively chargedpolyelectrolyte exposed in said second surface region contains a polymerrepeat unit having a zwitterion group. In another embodiment, the freemembrane or film on a substratum is characterized by a first surfaceregion and a second surface region have substantially planar exposedsurfaces wherein the first and second surface regions are substantiallycontiguous so as to define a pattern of regions having water contactangles that differ by at least 30 degrees.

Cytophilicity is a term used to describe whether a cell shows anaffinity for a surface, said affinity demonstrated by the adhesion ofthe cell to the surface and, at the extreme of affinity, by thespreading and differentiation of a cell into its biologically functionalform. The creation of surfaces having a range of cytophilicity, anobject of this invention, is accomplished using thin films ofpolyelectrolyte complex. Cytophilic surfaces are preferably preparedfrom polyelectrolyte complex comprising fluorinated polyelectrolytes.Cytophobic, or cell-repelling, surfaces are preferably prepared frompolyelectrolyte complex comprising zwitterionic and charged repeatunits.

It is known by those skilled in the art that cell adhesion and growth ona particular surface, including those surfaces comprising polymers,cannot be predicted a priori. For example, “So far, no generalprinciples that would allow prediction of the extent of attachment,spreading or growth of cultured cells on polymer surfaces have beenidentified” from W. M. Saltzman in “Principles of Tissue Engineering,”2nd Ed., Eds. R. P. Lanza, R. Langer, J. Vacanti, Academic Press, SanDiego, 2000. On the other hand, rough guidelines apply. For example,cell adhesion appears to be maximized on surfaces with intermediatewettability (See Y. Tamada & Y. Ikada, J. Biomed. Mater. Res. 28, 783(1994)). Fluorinated surfaces fall into the highly hydrophobic category,and thus the utility of fluorinated polyelectrolytes for cell adhesionand growth is unexpected. For example, Hyde et al. (J. Indust.Microbiol. Biotech. 19, 142 (1997)) describe the effectiveness offluorinated polymers at diminishing biofilm adherence to steel,polypropylene, and glass. The utility of fluorinated polyelectrolytes,which are distinctly non-biological materials, in promoting adhesion andgrowth of cells was unexpected. In particular, it could be reasoned thatthe positive polyelectrolyte might mimic the behavior of positivesurfactant-type molecules by disrupting the cell membrane andcompromising cell integrity. However, no evidence for dead cells on theperfluorinated surfaces could be found. Without being held to aparticular theory, it is possible that cells respond as they do to thehydrophobic nature of the perfluorinated surface, while the“fluorophobic” qualities of the cell membranes prevent them from mixingwith the multilayer components. Indeed, phase separations ofperfluorinated hydrocarbons from other (nonfluorinated) hydrophobicmolecules are common.

Preferred embodiments of this invention aid in precise control ofinterfacial solid/solution properties. Thus, in one preferred embodimentof this invention, a polyelectrolyte complex comprising at least onepositive and at least one negative polyelectrolyte, wherein at least oneof the polyelectrolytes is fluorinated, forms a film on the surface ofand in contact with a substrate. Preferably, the last-added, or “top”layer of said complex comprises fluorinated polyelectrolyte. Biologicalcells adhere, and grow on, said polyelectrolyte complex. Preferably, thefluid contacting said polyelectrolyte complex also contains essentialnutrients, buffer ions, and other chemical and biochemical species knownby those skilled in the art to promote the attachment, spreading,differentiation, and proliferation of said cells. Examples of growthmedia are provided below.

Preferred cells for growth on the polyelectrolyte complex comprisingfluorinated polyelectrolyte include smooth muscle cells, neuronal(nerve) cells, epithelial cells, and stem cells.

Preferred substrates on which the polyelectrolyte complex thin films ofthis invention are deposited include stents, catheters, vascular graftsand prostheses, ocular prostheses such as contact lenses and intraocularimplants, artificial valves for in vivo use, and other articlesimplanted either short-term or long-term in vivo, such as artificialorgans; dental implants, metal implants into bone, and metal objectsadapted for use in aqueous systems containing living organisms.

A further preferred substrate on which the polyelectrolyte complex thinfilms of this invention are deposited is a corneal implant. Preferredmaterials for said implant are hydroxyethylmethacrylate and copolymersthereof.

Further preferred substrates are cell and tissue culturing substrates,such as Petri dishes, roller bottles, microcarriers, porous structuralsupports for three dimensional cell growth, and similar cell and tissuegrowth templates.

A. Polyelectrolytes for Multilayer Films

The oppositely charged polymers (i.e., polyelectrolytes) used to formthe films are water and/or organic soluble and comprise one or moremonomer repeat units that are positively or negatively charged. Thepolyelectrolytes used in the present invention may be copolymers thathave a combination of charged and/or neutral monomers (e.g., positiveand neutral; negative and neutral; positive and negative; or positive,negative, and neutral). Regardless of the exact combination of chargedand neutral monomers, a polyelectrolyte of the present invention ispredominantly positively charged or predominantly negatively charged andhereinafter is referred to as a “positively-charged polyelectrolyte” ora “negatively-charged polyelectrolyte,” respectively.

Alternatively, the polyelectrolytes can be described in terms of theaverage charge per repeat unit in a polymer chain. For example, acopolymer composed of 100 neutral and 300 positively charged repeatunits has an average charge of 0.75 (3 out of 4 units, on average, arepositively charged). As another example, a polymer that has 100 neutral,100 negatively charged, and 300 positively charged repeat units wouldhave an average charge of 0.4 (100 negatively charged units cancel 100positively charged units leaving 200 positively charged units out of atotal of 500 units). Thus, a positively-charged polyelectrolyte has anaverage charge per repeat unit between 0 and 1 and a negatively-chargedpolyelectrolyte has an average charge per repeat unit between 0 and −1.An example of a positively-charged copolymer is PDADMA-co-PAC (i.e.,poly(diallyldimethylammonium chloride) and polyacrylamide copolymer) inwhich the PDADMA units have a charge of 1 and the PAC units are neutralso the average charge per repeat unit is less than 1.

Some polyelectrolytes comprise equal numbers of positive and negativerepeat units distributed throughout the polymer in a random,alternating, or block sequence. These polyelectrolytes are termed“amphiphilic” polyelectrolytes. For example, a polyelectrolyte moleculemay comprise 100 randomly distributed styrene sulfonate repeat units(negative) and 100 diallyldimethylammonium chloride repeat units(positive), said molecule having a net charge of zero.

Some polyelectrolytes comprise a repeat unit that has both a negativeand positive charge. Such repeat units are termed “zwitterionic” and thepolyelectrolyte is termed a “zwitterionic polyelectrolyte.” Thoughzwitterionic repeat units contribute equal number of positive andnegative repeat units, the zwitterionic group is still solvated andrelatively hydrophilic. An example of a zwitterionic repeat unit is3-[2-(acrylamido)-ethyldimethyl ammonio]propane sulfonate, AEDAPS.Zwitterion groups are present on polyelectrolytes as blocks or randomlydispersed throughout the polymer chain. Preferably, polyelectrolytescomprise at least about 15 mole % zwitterions repeat units, preferablybetween about 20 mole % and about 70 mole % zwitterion units, and morepreferably said polyelectrolytes comprise between about 30 mole % andabout 50 mole % zwitterion units. Preferred compositions ofpolyelectrolytes comprising zwitterionic repeat units also comprisebetween about 10% and about 90% non-zwitterionic charged repeat units.

The charges on a polyelectrolyte may be derived directly from themonomer units or they may be introduced by chemical reactions on aprecursor polymer. For example, PDADMA is made by polymerizingdiallyldimethylammonium chloride, a positively charged water solublevinyl monomer. PDADMA-co-PAC is made by the polymerization of a mixtureof diallyldimethylammonium chloride and acrylamide (a neutral monomerwhich remains neutral in the polymer). Poly(styrenesulfonic acid) isoften made by the sulfonation of neutral polystyrene.Poly(styrenesulfonic acid) can also be made by polymerizing thenegatively charged styrene sulfonate monomer. The chemical modificationof precursor polymers to produce charged polymers may be incomplete andtypically result in an average charge per repeat unit that is lessthan 1. For example, if only about 80% of the styrene repeat units ofpolystyrene are sulfonated, the resulting poly(styrenesulfonic acid) hasan average charge per repeat unit of about −0.8.

Examples of a negatively-charged synthetic polyelectrolyte includepolyelectrolytes comprising a sulfonate group (—SO₃ ⁻), such aspoly(styrenesulfonic acid) (PSS), poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS), sulfonated poly (ether ether ketone) (SPEEK),poly(ethylenesulfonic acid), poly(methacryloxyethylsulfonic acid), theirsalts, and copolymers thereof; and polycarboxylates such as poly(acrylicacid) (PAA) and poly(methacrylic acid).

Examples of a positively-charged synthetic polyelectrolyte includepolyelectrolytes comprising a quaternary ammonium group, such aspoly(diallyldimethylammonium chloride) (PDADMA),poly(vinylbenzyltrimethylammonium) (PVBTA), ionenes,poly(acryloxyethyltrimethyl ammonium chloride),poly(methacryloxy(2-hydroxy)propyltrimethyl ammonium chloride), andcopolymers thereof; polyelectrolytes comprising a pyridinium group suchas poly(N-methylvinylpyridinium) (PMVP), includingpoly(N-methyl-2-vinylpyridinium) (PM2VP), otherpoly(N-alkylvinylpyridines), and copolymers thereof; and protonatedpolyamines such as poly(allylaminehydrochloride) (PAH) andpolyethyleneimine (PEI).

Some synthetic polyelectrolytes used in accordance with the presentinvention generally become charged at certain pH values. For example,poly(acrylic acids) and derivatives begin to take on a negative chargewithin the range of about pH 4 to about 6 and are negatively charged athigher pH levels. Below this transition pH range, however, poly(acrylicacids) are protonated (i.e., uncharged). Similarly, polyamines andderivatives thereof take on a positive charge if the pH of the solutionis below their pK_(a). As such, and in accordance with the presentinvention, the pH of a polyelectrolyte solution may be adjusted by theaddition of an acid and/or base in order to attain, maintain, and/oradjust the electrical charge of a polyelectrolyte of a polyelectrolyteat the surface of, or within, a polyelectrolyte multilayer.

The state of ionization, or average charge per repeat unit, forpolyelectrolytes bearing pH-sensitive groups depends on the pH of thesolution. It is understood that the term “pH-sensitive,” as applied to afunctional group, refers to a functional group that exhibits differingdegrees of ionization over the working pH range of the experiment, while“pH-insensitive” refers to functional groups that maintain the samecharge (either positive or negative) over the working pH range of theexperiment. For example, a polyelectrolyte molecule comprising 100pH-insensitive positively charged units, such asdiallyldimethylammonium, DADMA, and 30 pH sensitive negatively chargedunits, such as acrylic acid, AA, will have a net charge of +100 at lowpH (where the AA units are neutral) and an average of +100/130 chargeper repeat unit; and a net charge of +70 at high pH (where 30 ionized AAunits cancel out 30 of the positive charges) and an average of +70/130charge per repeat unit. The different monomer units may be arrangedrandomly along the polymer chain (“random” copolymer) or they may existas blocks (“block” copolymer). The average charge per repeat unit isalso known as the “charge density.”

The molecular weight (number average) of synthetic polyelectrolytemolecules is typically about 1,000 to about 5,000,000 grams/mole,preferably about 10,000 to about 1,000,000 grams/mole. The molecularweight of naturally occurring polyelectrolyte molecules (i.e.,biomacromolecules), however, can reach as high as 10,000,000 grams/mole.The polyelectrolyte typically comprises about 0.01% to about 40% byweight of a polyelectrolyte solution, and preferably about 0.1% to about10% by weight.

The polyelectrolytes of the present invention comprise polymer chainbackbone units and pendant groups from the polymer chain backbone units.Polymer chain backbone units for use in the thin films of the presentinvention are preferably polyolefin groups (e.g., vinyl or allylgroups). Other polymer chain backbones units which may be applicableinclude polyamines, polyamides, polyethers, polyesters, polyimides,polysulfones, polyaryls, polyphenols, polyaramides, and copolymersthereof.

Many polyelectrolytes, such as PDADMA and PEI, exhibit some degree ofbranching. Branching may occur at random or at regular locations alongthe backbone of the polymer. For example, for the polymer repeat unitPDADMA, branching may occur due to the presence of two allyl groups onthe quaternary nitrogen. For PEI, branching may occur at secondarynitrogen groups along the polymer backbone. Branching may also occurfrom a central point and in such a case the polymer is referred to as a“star” polymer, if generally linear strands of polymer emanate from thecentral point. If, however, branching continues to propagate away fromthe central point, the polymer is referred to as a “dendritic” polymer.Branched polyelectrolytes, including star polymers, comb polymers, graftpolymers, and dendritic polymers, are also suitable for purposes of thisinvention.

Many of the foregoing polyelectrolytes have a very low toxicity. Infact, poly(diallyldimethylammonium chloride),poly(2-acrylamido-2-methyl-1-propane sulfonic acid), and theircopolymers are used in the personal care industry, e.g., in shampoos.Also, because the polyelectrolytes used in the method of the presentinvention are synthetic or synthetically modified natural polymers,their properties (e.g., charge density, viscosity, water solubility, andresponse to pH) may be tailored by adjusting their composition.

By definition, a polyelectrolyte solution comprises a solvent. Anappropriate solvent is one in which the selected polyelectrolyte issoluble. Thus, the appropriate solvent is dependent upon whether thepolyelectrolyte is considered to be hydrophobic or hydrophilic. Ahydrophobic polymer displays a less favorable interaction energy withwater than a hydrophilic polymer. While a hydrophilic polymer is watersoluble, a hydrophobic polymer may only be sparingly soluble in water,or, more likely, insoluble in water. Likewise, a hydrophobic polymer ismore likely to be soluble in organic solvents than a hydrophilicpolymer. In general, the higher the carbon to charge ratio of thepolymer, the more hydrophobic it tends to be. For example, polyvinylpyridine alkylated with a methyl group (PNMVP) is considered to behydrophilic, whereas polyvinyl pyridine alkylated with an octyl group(PNOVP) is considered to be hydrophobic. Thus, water is preferably usedas the solvent for hydrophilic polyelectrolytes, and organic solventssuch as ethanol, methanol, dimethylformamide, acetonitrile, carbontetrachloride, and methylene chloride are preferably used forhydrophobic polyelectrolytes. Preferred solvents for fluorinatedpolymers are themselves fluorinated. Another preferred solvent forfluorinated polymers is supercritical carbon dioxide. Since somesolvents are known to be incompatible with some plastic materials,preferred solvents for depositing polyelectrolyte complex thin films onplastics are water and alcohols.

Examples of polyelectrolytes that are soluble in water includepoly(styrenesulfonic acid), poly(2-acrylamido-2-methyl-1-propanesulfonic acid), sulfonated lignin, poly(ethylenesulfonic acid),poly(methacryloxyethylsulfonic acid), poly(acrylic acids),poly(methacrylic acids), their salts, and copolymers thereof; as well aspoly(diallyldimethylammonium chloride),poly(vinylbenzyltrimethylammonium), ionenes, poly(acryloxyethyltrimethylammonium chloride), poly(methacryloxy(2-hydroxy)propyltrimethyl ammoniumchloride), and copolymers thereof; polyelectrolytes comprising apyridinium group, such as, poly(N-methylvinylpyridium); and protonatedpolyamines, such as, poly(allylamine hydrochloride) andpoly(ethyleneimine).

Examples of polyelectrolytes that are soluble in non-aqueous solvents,such as ethanol, methanol, dimethylformamide, acetonitrile, carbontetrachloride, and methylene chloride includepoly(N-alkylvinylpyridines), and copolymers thereof in which the alkylgroup is longer than about 4 carbon atoms. Other examples ofpolyelectrolytes soluble in organic solvents includepoly(styrenesulfonic acid), poly(diallyldimethylammonium chloride),poly(N-methylvinylpyridinium), and poly(ethyleneimine) where the smallpolymer counterion, such as chloride or bromide, has been replaced by alarge hydrophobic counterion such as tetrabutyl ammonium, tetraethylammonium, iodine, hexafluorophosphate, tetrafluoroborate, ortrifluoromethane sulfonate. Preferred counterions that assist thesolubility of polyelectrolytes in organic solvents of low dielectricconstant, and in supercritical CO₂, include perfluorinated alkanesulfonates, preferably of length from 3 to 10 carbons, andperfluoroalkane carboxylic acids, preferably of length from 3 to 18carbons.

In some applications, the PEMU preferably inhibits cell adhesion andgrowth. Substrates for which it is advantageous to inhibit cell adhesionand growth include ocular prostheses such as contact lenses andintraocular lenses, vascular grafts and prostheses, artificial organs,and stents. In order to minimize adhesion of cells on articles implantedin vivo, preferred polyelectrolyte complex coatings for inhibiting celladhesions on articles comprise polyelectrolyte molecules comprisingzwitterion repeat units and net charged repeat units.

Preferred net charged repeat units include sulfonates,styrenesulfonates, 2-acrylamido-2-methyl-1-propane sulfonic acid,ethylenesulfonic acid, methacryloxyethylsulfonic acid, sulfonated etherether ketone, diallyldialkylammonium, vinylbenzyltrimethylammonium,ionenes, acryloxyethyltrimethyl ammonium chloride,methacryloxy(2-hydroxy)propyltrimethyl ammonium,N-methylvinylpyridinium, other N-alkylvinyl pyridiniums, N-aryl vinylpyridiniums, alkyl- or aryl imidazolium, carboxylates such as acrylicacid and methacrylic acid, phosphates, protonated pyridines, protonatedimidazoles, and protonated primary, secondary, or tertiary amines. TableI below depicts the names and structures of net charged repeat unitswhich may be incorporated into co-polymers also comprising zwitterionicrepeat units.

TABLE I Net Charged Repeat Units for use in Polyelectrolytes NameStructure Diallyldimethylammonium (PDADMA)

Styrenesulfonic acid (PSS)

Acrylic acid (PAA)

Allylamine (PAH)

N-methyl-2-vinylpyridinium (PM2VP)

Methacrylic acid (PMA)

Examples of zwitterionic repeat units includeN,N-dimethyl-N-acryloyloxyethyl-N-(3-sulfopropyl)-ammonium betaine,N,N-dimethyl-N-acrylamidopropyl-N-(2-carboxymethyl)-ammonium betaine,N,N-dimethyl-N-acrylamidopropyl-N-(3-sulfopropyl)-ammonium betaine,N,N-dimethyl-N-acrylamidopropyl-N-(2-carboxymethyl)-ammonium betaine,2-(methylthio)ethyl methacryloyl-S-(sulfopropyl)-sulfonium betaine,2-[(2-acryloylethyl)dimethylammonio]ethyl 2-methyl phosphate,2-(acryloyloxyethyl)-2′-(trimethylammonium)ethyl phosphate,[(2-acryloylethyl)dimethylammonio]methyl phosphonic acid,2-methacryloyloxyethyl phosphorylcholine (MPC),2-[(3-acrylamidopropyl)dimethylammonio]ethyl 2′-isopropyl phosphate(AAPI), 1-vinyl-3-(3-sulfopropyl)imidazolium hydroxide,(2-acryloxyethyl) carboxymethyl methylsulfonium chloride,1-(3-sulfopropyl)-2-vinylpyridinium betaine,N-(4-sulfobutyl)-N-methyl-N,N-diallylamine ammonium betaine (MDABS),N,N-diallyl-N-methyl-N-(2-sulfoethyl) ammonium betaine,N,N-dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl) ammoniumbetaine, N,N-dimethyl-N-acryloyloxyethyl-N-(3-sulfopropyl)-ammoniumbetaine, N,N-dimethyl-N-acrylamidopropyl-N-(2-carboxymethyl)-ammoniumbetaine, N,N-dimethyl-N-methacryloyloxyethyl-N-(3-sulfopropyl)-ammoniumbetaine, and N,N-dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl)ammonium betaine.

More preferably, said polyelectrolyte complexes comprise both carboxylicacid and zwitterionic repeat units. Preferably, the zwitterionic andcarboxylic acid groups are on the same polyelectrolyte molecule, orcopolyelectrolyte. The preferred zwitterion content on saidcopolyelectrolytes is at least about 15 mole percent, preferably betweenabout 20 mole percent and about 70 mole percent, more preferably betweenabout 30 mole percent and about 50 mole percent, with carboxylic acidunits preferably comprising the balance of the copolyelectrolyte.

Preferred zwitterionic repeat units include3-[2-(acrylamido)-ethyldimethyl ammonio]propane sulfonates (AEDAPS).Preferred copolymers comprising a net charged repeat units andzwitterionic repeat units include poly(acrylicacid)-co-poly(3-[2-(acrylamido)-ethyldimethyl ammonio]propanesulfonate), PAA-co-PAEDAPS. Since zwitterion groups are net chargeneutral, they interact only weakly with charged groups. Thus, thepreferred amount of zwitterion repeat unit should be not so high thatthe copolymer does not form complexes with oppositely-chargedpolyelectrolyte. The maximum percentage of zwitterion unit that can betolerated depends on the chemical identity of the net charged unit thatis holding the polyelectrolyte complex together. Preferred molepercentages of zwitterion do not exceed about 80 mole %, unlessprovision for chemical crosslinking is made during or after multilayerformation. The polymer repeat unit having a zwitterion group ispreferably located on a polyelectrolyte molecule that also comprises anet charged repeat unit, preferably a carboxylic acid, preferablyacrylic acid. Table II shows the structures of preferred zwitterionicrepeat units for use in membranes for inhibiting cell adhesion.

TABLE II Zwitterion Repeat Units for use in Polyelelectrolytes NameStructure 3-[2-(acrylamido)-ethyl- dimethyl ammonio]pro- pane sulfonate(AEDAPS)

N-propane sulfonate-2-vinyl-pyridine (P2PSVP)

Optionally, the polyelectrolytes comprise an uncharged repeat unit thatis preferably hydrophilic. Preferred uncharged hydrophilic repeat unitsare acrylamide, vinyl pyrrolidone, ethylene oxide, and vinylcaprolactam. The structures of exemplary uncharged repeat units areshown in Table III.

TABLE III Uncharged Repeat Units for use in Polyelectrolytes NameStructure Acrylamide

Vinylpyrrolidone

Ethylene Oxide

Vinylcaprolactam

In order to further inhibit growth of cells that may have attached toarticles coated with films of polyelectrolyte complexes comprisingcopolyelectrolytes of zwitterionic and carboxylic acid repeat units,said films preferably further comprise paclitaxel or sirolimus or otheragents known to those skilled in the art to inhibit cell growth andproliferation. Substrates for which it is advantageous to include agentsknown to inhibit cell growth and proliferation include stents,catheters, vascular grafts, vascular prostheses, contact lenses,intraocular implants, artificial valves for in vivo use, and artificialorgans. Preferably, the film of polyelectrolyte complex comprisingzwitterionic repeat units (hydrophilic) is deposited on a hydrophobicstratum, said hydrophobic stratum enhancing the adhesion of thehydrophilic stratum to the substrate and also enhancing the uptake ofagents such as paclitaxel and sirolimus. Since these agents arerelatively hydrophobic, they will partition more strongly intohydrophobic strata.

In another preferred embodiment of this invention, the surface of asubstrate is coated with a thin film of polyelectrolyte complexcomprising a zwitterionic repeat unit, and the coating is used toprevent or reduce the adsorption of platelets, bacteria, and marinemicroorganisms. Films which reduce the adsorption of platelets areadvantageous for coating stents. Anti-microbial films are advantageousfor coating contact lenses, intraocular lenses, vascular grafts,catheters, artificial organs, and stents.

Films for which it is advantageous to reduce the adsorption of marineorganisms are useful for coating metal surfaces adapted for use inaqueous systems having living organisms. Such substrates that areroutinely exposed to water include, for example, the hull of a ship. Inthese applications, the highly hydrophilic polyelectrolytes havingpolymer repeat units having zwitterion groups may enhance corrosion ofthe metal substrate. Therefore, it is preferred to deposit a firststratum of polyelectrolyte complex comprising polymers havingfluorinated repeat units onto the surface of the metal substrate tocontrol and thereby enhance the hydrophobicity of the metal substrate,which additionally imparts corrosion resistance. After the first stratumof polyelectrolyte complex comprising fluorinated repeat units isdeposited, a second stratum of polyelectrolyte complex comprisingpolymers having zwitterion groups may be deposited to inhibit theadsorption of marine organisms.

In some applications, the polyelectrolyte film preferably enhances celladhesion, growth, and in some embodiments differentiation. Substratesfor which it is advantageous to enhance cell adhesion, growth, anddifferentiation include vascular grafts and catheters where the growthof endothelial cells on the surface of the graft or catheter renders thegraft or catheter biocompatible, dental implants, cell and tissueculturing substrates, and metal implants into bone. Preferredpolyelectrolyte film coatings for inducing cell adhesion and growth oncomplex objects comprise fluorinated polymers.

The fluorinated polyelectrolytes comprising the polyelectrolyte thinfilms of this invention are preferably copolymers, orcopolyelectrolytes, comprising fluorinated and non-fluorinated repeatunits. Said repeat units may be disposed in a random or block fashion onthe backbone of said copolyelectrolytes. Preferred fluorinatedcopolyelectrolytes comprise charged non-fluorinated with nonchargedfluorinated repeat units, or charged fluorinated with nonchargednonfluorinated repeat units. Other preferred fluorinatedpolyelectrolytes comprise charged fluorinated repeat units with chargednonfluorinated repeat units. Fluorinated copolyelectrolytes arepreferably made by post-polymerization reactions on polymers, such asalkylation, or by polymerization of fluorinated monomers or mixtures offluorinated monomers. Mole percentages of fluorinated repeat units onfluorinated copolyelectrolytes are preferably from 10% to 95%, and morepreferably from 20% to 95%.

The fluorinated polyelectrolytes of the present invention arehydrophobic, preferably having water/air/surface interfacial contactangles greater than about 70 degrees, more preferably greater than about80 degrees, and even more preferably greater than about 90 degrees.Measurement of the interfacial contact angle between a water surface anda coated surface is a well known method of assessing the wettingproperties of water on a material (see R. J. Good, J. Adhesion Sci.Technol., 12, 1269, (1992)). If the contact angle of water on a coatingis low, the surface is said to be hydrophilic. If the contact angle ishigh, the surface is said to be hydrophobic. Surfaces with contactangles of greater than 90 degrees are particularly effective forantiwetting applications. The contact angle of water on polyelectrolytemultilayers depends on the combination of polyelectrolytes and also onwhich polyelectrolyte is used for the “top” layer (see for example Chenand McCarthy, Macromolecules, 30, 78 1997; and Yoo et al.Macromolecules, 31, 4309 (1998)).

Preferred fluorinated polyelectrolytes are either positive or negative.A range of repeat units may be included in the predominantly positivelycharged polymer, the predominantly negatively charged polymer, or both.In one embodiment, the repeat unit is a positively charged repeat unitcomprising groups selected from the group consisting of a quaternarynitrogen atom (N⁺), a sulfonium (S⁺) atom, or a phosphonium atom (P⁺).Thus, for example, the quaternary nitrogen may be part of a quaternaryammonium moiety (—N⁺R_(a)R_(b)R_(c) wherein R_(a), R_(b), and R_(c) areindependently alkyl, aryl, or mixed alkyl and aryl), a pyridiniummoiety, a bipyridinium moiety, or an imidazolium moiety, the sulfoniumatom may be part of a sulfonium moiety (—S⁺R_(d)R_(e) wherein R_(d) andR_(e) are independently alkyl, aryl, or mixed alkyl and aryl) and thephosphonium atom may be part of a phosphonium moiety (—P⁺R_(f)R_(g)R_(h)wherein R_(f), R_(g), and R_(h) are independently alkyl, aryl, or mixedalkyl and aryl). In another embodiment, the repeat unit is a negativelycharged repeat unit comprising groups selected from the group consistingof sulfonates (—SO₃ ⁻), phosphates (—OPO₃ ⁻), or sulfates (—SO₄ ⁻). Forillustrative purposes, certain of these moieties are shown as vinylrepeat units:

Vinyl Polymer Repeat Unit

-   -   wherein R₁, R₂, and R₃ are each independently: —(CH₂)_(m)H or        —(CH_(x)F_(2-x))_(n)F and m and n are independently 0 to 12, x        is 0, 1, or 2 and V is a group selected from among the        following:    -   fluorinated hydrocarbons having the structure:        —(CH₂)_(p)(CF₂)_(q)F, —(CH₂)_(p)(CF₂)_(q)COOH,        —(CH₂)_(p)(CF₂)_(q)OPO₃ ⁻, —(CH₂)_(p)(CF₂)_(q)SO₃ ⁻,        —(CH₂)_(p)(CF₂)_(q)OSO₃ ⁻, —O(CH₂)_(p)(CF₂)_(q)F,        —O(CH₂)_(p)(CF₂)_(q)SO₃ ⁻ and wherein p is 0 to 6 and q is 1 to        21;    -   fluorinated amides having the structure:

wherein R₄ is —(CH₂)_(p)(CF₂)_(q)F, —(CH₂)_(p)(CF₂)_(q)COOH,—(CH₂)_(p)(CF₂)_(q)OPO₃ ⁻, —(CH₂)_(p)(CF₂)_(q)SO₃ ⁻,—(CH₂)_(p)(CF₂)_(q)OSO₃ ⁻ and wherein p is 0 to 6 and q is 1 to 21;

-   -   fluorinated esters having the structure:

wherein R₅ is —(CH₂)_(p)(CF₂)_(q)F, —(CH₂)_(p)(CF₂)_(q)COOH,—(CH₂)_(p)(CF₂)_(q)OPO₃ ⁻, —(CH₂)_(p)(CF₂)_(q)SO₃ ⁻,—(CH₂)_(p)(CF₂)_(q)OSO₃ ⁻ and wherein p is 0 to 6 and q is 1 to 21;

-   -   fluorinated phenyl groups having the structure:

wherein n is 2 to 5; or

wherein R₆ is=—(CH₂)_(p)(CF₂)_(q)F or —O(CH₂)_(p)(CF₂)_(q)F and whereinp is 0 to 6 and q is 1 to 21;

-   -   fluorinated pyridiniums having the structure:

wherein R₇ is —(CH₂)_(p)(CF₂)_(q)F and wherein p is 0 to 6 and q is 1 to21;

-   -   fluorinated imidazoliums having the structure:

wherein R₈ is —(CH₂)_(p)(CF₂)_(q)F and wherein p is 0 to 6 and q is 1 to21;

-   -   fluorinated quaternary nitrogens having the structure:

wherein R₉, R₁₀, and R₁₁ are each independently —(CH₂)_(p)(CF₂)_(q)Fwherein p is 0 to 6 and q is 1 to 21 or -arylF_(z) wherein z is 2 to 8;

-   -   fluorinated sulfoniums having the structure:

wherein R₁₂ and R₁₃ are each independently —(CH₂)_(p)(CF₂)_(q)F whereinp is 0 to 6 and q is 1 to 21 or -arylF_(z) wherein z is 2 to 8; and

-   -   fluorinated phosphoniums having the structure:

wherein R₁₄, R₁₅, and R₁₆ are each independently —(CH₂)_(p)(CF₂)_(q)Fwherein p is 0 to 6 and q is 1 to 21 or -arylF_(z) where z=2 to 8.

For illustrative purposes, certain of these moieties are shown as allylrepeat units (e.g., PDADMA):

wherein R₂₁ and R₂₂ are —(CH₂)_(p)(CF₂)_(q)F, wherein p and q areindependently selected for R₂₁ and R₂₂, and p is 0 to 6 and q is 1 to21.

The positive fluorinated polyelectrolytes of this invention arepreferably prepared by the alkylation of a nitrogen group, a sulfurgroup, or a phosphorus group by an alkylating molecule comprising two ormore fluorine atoms. Said alkylating molecule also comprises a groupthat may be displaced on reaction (a “leaving group”) that is well knownto those skilled in the art. Examples of preferred leaving groups arechloride, bromide, iodide, and toluene sulfonate. Preferrednitrogen-containing groups on polymers to be alkylated are the pyridinegroup, imidazoles, and primary, secondary, or tertiary amines.Advantageously, alkylation often proceeds with the simultaneous creationof a positive charge. For efficiency of alkylation, preferredfluorinated hydrocarbons have one or two carbons that do not bearfluorines next to the leaving group.

Preferably, fluorinated copolyelectrolytes comprising chargedfluorinated groups and charged unfluorinated groups are formed by thealkylation of residual nitrogen groups, sulfur groups, or phosphorusgroups that were not fluorinated by the fluorinated alkylating agent.Alkylation reactions with fluorinated molecules are incomplete,typically reaching yields of less than 100%, typically about 50%. As aresult, a fraction of the nitrogen, sulfur, or phosphorous groups arepositively charged and comprise fluorinated hydrocarbons, while theremaining fraction is uncharged. Advantageously, the degree of chargecan be controlled and increased by further alkylating the residualnitrogen groups, preferably with saturated non-fluorinated hydrocarbonscomprising a leaving group as is known to those skilled in the art.Preferably, alkylation with the fluorinated molecules occurs beforealkylation with the saturated hydrocarbons.

Preferred uncharged fluorinated monomers include fluorovinyl ethers,such as CF₂═CF(OC₂F₄)_(n)—R where n is from 1 to 12 and R is a hydroxyl;alkoxy; aryl; or alkyl group, fluorinated styrenes, fluorinated olefins,vinylperfluoroesters, and vinylperfluoracrylates.

Preferred anionic fluorinated polyelectrolytes comprise the sulfonategroup. Preferred anionic fluorinated polyelectrolytes comprising thesulfonate group are poly perfluorinated sulfonated ionomers including apolymer marketed under the trade name Nafion™ and sulfonatedperfluorinated alkylvinyl vinyl ethers. Other preferred anionicpolyelectrolytes comprise perfluorinated vinyl carboxylic acids. TableIV shows the structures of fluorinated polyelectrolytes for using inbuilding PEMUs of the present invention.

TABLE IV Fluorinated Polyelectrolyte Repeat Units for use in FluorinatedPolyelectrolytes Name Structure 4-vinyl-trideca-fluoro-octyl pyridiniumiodide-co-4-vinyl pyridine (PFPVP)

NAFION

Preferred charged nonfluorinated polyelectrolyte repeat units includesulfonates, styrenesulfonates, 2-acrylamido-2-methyl-1-propane sulfonicacid, ethylenesulfonic acid, methacryloxyethylsulfonic acid, sulfonatedether ether ketone, diallyldialkylammonium,vinylbenzyltrimethylammonium, ionenes, acryloxyethyltrimethyl ammoniumchloride, methacryloxy(2-hydroxy)propyltrimethyl ammonium,N-methylvinylpyridinium, other N-alkylvinyl pyridiniums, N-aryl vinylpyridiniums, alkyl- or aryl imidazolium, protonated pyridines,protonated imidazoles, and protonated primary, secondary, or tertiaryamines. More preferred nonfluorinated charged repeat units includecarboxylates such as acrylic acid and methacrylic acid, vinylphosphates, and vinylphospholipids.

Table V below depicts the names and structures of repeat units which maybe incorporated as uncharged or charged, fluorinated or unfluorinatedrepeat units in the polyelectrolytes for use in building the thin filmsof the present invention.

TABLE V Repeat Units for use in Fluorinated Polyelectrolytes Name ofBase Structure Unalkylated Repeat Unit Fluorinated Alkylated Repeat UnitUnfluorinated Alkylated Repeat Unit Diallyl ammonium (PDADMA)

Styrene sulfonic acid (PSS)

Allyl amine (PAH)

Vinyl pyridine (PVP)

Dialkyl amino Ethyl acrylamido

In order to enhance the adhesion of polyelectrolyte complex thin filmscomprising fluorinated polyelectrolyte to a substrate, an intermediatenonfluorinated polyelectrolyte layer, or a stratum of nonfluorinatedpolyelectrolyte complex, is placed between said thin film and thesubstrate. Preferred polyelectrolytes for this intermediate stratum orlayer include polyethyleneimine and poly(N-alkylvinylpyridiniums), wherethe N-alkyl group comprises 4 to 18 carbons. Saidpoly(N-alkylvinylpyridiniums) are organic-soluble and, having ahydrophobic character intermediate between a water-solublepolyelectrolyte and a fluorinated polyelectrolyte, enhance the adhesionof the fluorinated polyelectrolyte to a substrate.

It is known by those skilled in the art that the top, or outer, layer ofa polyelectrolyte layer has the most effect on surface hydrophobicity.Accordingly, in one embodiment of this invention, the initial layers 0through n of a multilayer are prepared from nonfluorinatedpolyelectrolytes, preferably those listed above, and the n+1 and n+2layers comprise fluorinated positive polyelectrolyte and fluorinatednegative polyelectrolyte. The use of fluorinated polyelectrolytes inonly the top layers conserves potentially costly materials.

In another embodiment of this invention, the initial layers 0 through nof a multilayer are prepared from nonfluorinated polyelectrolytes,preferably those listed above, and the n+1 layer comprises fluorinatedpositive polyelectrolyte, preferably PFPVP. The contact angle of wateron a PFPVP surface is higher than the contact angle on a Nafion surface.Therefore, a single layer of PFPVP is advantageously more hydrophobicthan a single layer of Nafion.

In one embodiment of this invention, an article to be implanted in vivois first coated with a film of polyelectrolyte complex comprisingfluorinated polyelectrolyte, the outer layer of said film preferablycomprising fluorinated polyelectrolyte, and then the article is coatedwith a layer of protein found in the animal into which the article is tobe implanted. Preferably, said protein is serum albumin. A coating ofserum albumin will render the article more biocompatible.

In yet another preferred embodiment of this invention, a thin film ofpolyelectrolyte complex comprising fluorinated repeat units alsocomprises polynucleic acids, such as DNA and RNA. Cells are thencultured onto this surface and the nucleic acid material preferablytransfects into the cell, modifying the genetic material of said cell.The genes corresponding to the polynucleic acid are preferably expressedby the cell, leading to the production of select proteins. Preferably,the polynucleic acid is adsorbed to the surface of the polyelectrolytecomplex film comprising fluorinated polyelectrolyte. More preferably, acomplex of the polynucleic acid and a positively charged fluorinatedpolyelectrolyte are formed on the surface of a stamp and said complex istransferred directly to a surface by stamping. A positive fluorinatedpolyelectrolyte is preferred over a negative fluorinated polyelectrolytebecause genetic material (i.e., DNA and RNA) is negatively charged andwill therefore complex with positively charged polyelectrolyte.Optionally, the fluorinated polyelectrolyte complex film comprisingpolynucleic acid is coated on the surface of a scaffold, preferablycomprising one of the biodegradable polymers listed below. Said coatedscaffold encourages simultaneously the growth of cells and thetransfection of genetic material from the scaffold to the growing cells.

To assist in maintaining the physical integrity of the polyelectrolytethin film, in one preferred embodiment a small amount of chemicalcrosslinking is introduced into the film. Crosslinking is preferablyaccomplished by including difunctional monomers in the polyelectrolytescomprising the thin film. For example, a divinyl repeat unit added tothe polymerization reaction will be incorporated into twopolyelectrolyte chains, giving a crosslink at the connection point.Alternatively, a polyelectrolyte film may be treated with a difunctionalcrosslinking agent. A preferred crosslinking agent is a dihalogenatedcompound, such as an aromatic or aliphatic dibromide, which is able toalkylate residual unalkylated units on two adjoining polyelectrolytechains. Another preferred method of crosslinking a formedpolyelectrolyte thin film is heat treatment. For example, Dai et al.(Langmuir 17, 931 (2001)) disclose a method of forming amide crosslinksby heating a polyelectrolyte multilayer comprising amine and carboxylicacid groups. Yet another preferred method of introducing crosslinking,disclosed by Kozlovskaya et al. (Macromolecules, 36, 8590 (2003)) is bythe addition of a carbodiimide, which activates chemical crosslinking.The level of crosslinking is preferably 0.01% to 50%, and morepreferably 0.1% to 10%.

B. Additives for Use in Building PEMUs

The PEMUs of the present invention may be built by incorporatingadditives in the polyelectrolyte solutions which affect the thin filmmechanical properties.

Optionally, the polyelectrolyte solutions may comprise one or more“salts.” A “salt” is defined as a soluble, ionic, inorganic compoundthat dissociates to stable ions (e.g., sodium chloride). A salt isincluded in the polyelectrolyte solutions to control the thickness ofthe adsorbed layers. More specifically, including a salt increases thethickness of the adsorbed polyelectrolyte layer. In general, increasingthe salt concentration increases the thickness of the layer for a givenpolyelectrolyte concentration and contact time. This phenomenon islimited, however, by the fact that upon reaching a sufficient saltconcentration, multilayers tend to dissociate. Typically, the amount ofsalt added to the polyelectrolyte solution is about 10% by weight orless.

Both dip coating and spraying permit a wide variety of additives to beincorporated into a film as it is formed. Additives that may beincorporated into polyelectrolyte multilayers include inorganicmaterials such as metallic oxide particles (e.g., silicon dioxide,aluminum oxide, titanium dioxide, iron oxide, zirconium oxide, andvanadium oxide) and clay minerals (e.g., hectorite, kaolin, laponite,montmorillonite, and attapulgite). These particles typically range insize from about 1 nanometer to about 10 micrometers. For example,nanoparticles of zirconium oxide added to a polyelectrolyte solution orcomplex solution tend to improve the abrasion resistance of thedeposited film. See Rosidian et al., Ionic Self-assembly of Ultra HardZrO ₂ /polymer Nanocomposite Films, Advanced Materials 10, 1087-1091(1998).

Other additives include carbon fibers and carbon nanotubes (having adiameter less than 100 nanometer and an aspect ratio (length to width)of at least 10:1). Optionally, charged Teflon™ particles may beincorporated into the thin films. Typically, Teflon™ particles arecharge neutral, but surfactants may be added onto the surface of theparticles to impart a charge which may be positive or negative dependingupon the surfactant employed. Particle additives are added to thepolyelectrolyte solutions, or are layered between polyelectrolyte layersin separate coating steps.

Optionally, the polymer film further comprises agents known to promotecell growth, i.e., growth factors, and other cell nutrients. Optionally,the film further comprises agents known to inhibit cell growth, i.e.,paclitaxel or sirolimus.

C. Deposition Methods and Substrates

While this invention employs polyelectrolyte complex thin films, apreferred method of depositing said complex is by the alternatinglayer-by-layer deposition method. The preferred method of alternatingexposure of the substrate or material to be coated is by alternateimmersion in polyelectrolyte solutions, or alternate spraying ofpolyelectrolyte solutions. The alternating polyelectrolyte layeringmethod, however, does not generally result in a layered morphology ofthe polymers with the film. Rather, the polymeric componentsinterdiffuse and mix on a molecular level upon incorporation into thethin film. See Lösche et al., Macromolecules 31, 8893 (1998). Thus, thepolymeric components form a true molecular blend, referred to as a“polyelectrolyte complex,” with intimate contact between polymers drivenby the multiple electrostatic complexation between positive and negativepolymer segments. The complexed polyelectrolytes within the PEMU filmhave similar morphology to a polyelectrolyte complex formed by mixingsolutions of positive and negative polyelectrolyte. It is also knownthat although there is extensive intermingling of neighboring layersover a range of 4-6 nominal layers, it is possible to obtain actuallayers of different composition, or strata, by interspersing severallayers made from one pair of polyelectrolytes by several layers madefrom a different pair. See Lösche et al., Macromolecules 31, 8893(1998). For example, if polymers A and C are positively charged andpolymers B and D are negatively charged, about 3 or 4 pairs of A/Blayers followed by about 3 or 4 pairs of A/D or C/D layers will producetwo strata of distinct composition. The preferred concentration forsolutions comprising polyelectrolytes to be deposited is in the range0.01 weight % to 10 weight %, and preferably 0.1 weight % to 1 weight %.

Alternatively, the thin film coating may be applied to a surface using apre-formed polyelectrolyte complex. See Michaels, PolyelectrolyteComplexes, Ind. Eng. Chem. 57, 32-40 (1965) and Michaels (U.S. Pat. No.3,467,604). This is accomplished by mixing the oppositely-chargedpolyelectrolytes to form a polyelectrolyte complex precipitate which isthen dissolved or re-suspended in a suitable solvent/liquid to form apolyelectrolyte complex solution/dispersion. The polyelectrolyte complexsolution/dispersion is then applied to the substrate surface and thesolvent/liquid is evaporated, leaving behind a film comprising thepolyelectrolyte complex. To aid in dissolution or dispersion of thecomplex, both a salt, such as sodium bromide, and an organic solvent,such as acetone is optionally added to the solution comprising theprecipitated complex. It is known that the material obtained by layeringtwo polyelectrolytes is substantially the same composition as materialobtained by mixing and precipitating said polymers to form apolyelectrolyte complex.

In one embodiment of this invention, the polyelectrolyte complex isformed on a polymer or plastic surface. Polyelectrolyte complexes,especially those formed by the layer-by-layer alternating depositiontechnique, are known by those skilled in the art to adhere to plasticmaterials. For example, Chen and McCarthy (Macromolecules, 30, 78 (1997)describe the layer-by-layer deposition of polyelectrolyte complex onpoly(ethylene terephthalate). Even fluorinated polymers, such asDupont's Teflon™, are known to be coated by polyelectrolyte complexusing the layer-by-layer technique (see Hsieh et al. Macromolecules, 30,8453 (1997). Barker et al. (Anal. Chem., 72, 5925 (2000)) (See alsoLocascio et al. U.S. Pat. Pub. No. 2002/0053514) have disclosed thelayer-by-layer deposition of polyelectrolytes on plastic microfluidicchannels. Thus, preferred plastic substrates on which polyelectrolytecomplex films may be formed include polycarbonate, poly(methylmethacrylate), polystyrene, poly(ethylene terephthalate), polysulfone,or polyamide, with the proviso that solvents used to process thefluorinated polyelectrolyte complex thin film does not attack thesubstrate on which the thin film of complex is being formed.

For fast throughput and coating of surfaces, one method of applying thepolyelectrolyte complex is by spraying of a surface. Spraying isespecially preferred when applying the coating to large areas usingalternating exposure of oppositely-charged polyelectrolyte solutions.Spraying alternating oppositely-charged polyelectrolyte solutions hasseveral advantages over the Michaels coating and evaporation method,including: improved control over film thickness especially the abilityto make extremely thin films (e.g., less than about 1 μm), and enhanceduniformity of film thickness especially over uneven surfaces andcontours. The solutions may be sprayed onto a substrate by anyapplicable means (e.g., an atomizer, an aspirator, ultrasonic vaporgenerator, entrainment in compressed gas, or inkjet sprayer). In fact, ahand operated “plant mister” has been used to spray the polyelectrolytesolutions. Typically, the droplet size in the spray is about 10 nm toabout 1 mm in diameter. Preferably, the droplet size is about 10 μm to100 μm in diameter. The coverage of the spray is typically about 0.001to 1 mL/cm², and preferably about 0.01 to 0.1 mL/cm².

In order to create a pattern of polyelectrolyte on a surface, sprayingis preferably done though a mask which defines the pattern. Preferably,spraying through a mask is performed with a fine spray, such as thatproduced by ultrasonic vaporization. The mask is preferably placed on ornear the surface to be coated. Many patterns of different levels ofcomplexity are possible. Preferred dimensions for features on the maskrange from about 10 micrometers to several centimeters.

It is known to those skilled in the art that fluorinated groups,especially chains of fluorinated hydrocarbons, cause aggregation,especially in aqueous solution. A system comprising aggregates as smallparticles dispersed in a solvent is known as a dispersion, or asuspension. A suspension of particles that are small enough such thatthey do not settle out is known as a colloid. Colloids in aqueoussolution are often stabilized against aggregation into larger particlesby having a surface charge. The surface charge can be derived directlyfrom the material forming the colloid, or it can be maintained by theadsorption of a surface active agent, or surfactant. Charged surfactantsstabilize suspensions by causing the surface of the particles to havethe same charge and therefore repel each other. Neutral surfactants relyon steric interactions (repulsions) to prevent aggregation of suspendedparticles. In one embodiment of this invention, at least one of thefluorinated polymers is dispersed as a quasi-stable suspension in asolvent, and said suspensions are employed in the multilayering process.Preferably, the solvent comprising such suspensions comprises water. Theparticle size of the suspension is preferably less than about onemicrometer, and more preferably less than about 100 nanometers.Preferably the particles comprising said suspension or dispersion bear anet surface charge.

Preferred suspensions comprise fluorinated polymers comprising chargedrepeat units. Other preferred suspensions comprise telomerizedfluoropolymers, including those produced by Asahi Glass, Atochem,Daikin, such as Daikin 3310 or 3311, Dupont, such as Dupont Tufcoat(Anionic), and Clariant, such as the NUVA fluoropolymers, such as NUVACPA, NUVA 5006, and Peach State Labs, such as Myafax WS.

It is also known to those skilled in the art that fluoropolymers may bedissolved or dispersed in supercritical carbon dioxide, CO₂. Thedielectric constant of supercritical CO₂ is low and matches that offluorinated hydrocarbons including fluorinated monomers, which may bepolymerized to yield fluorinated polymers in supercritical CO₂ (seeDeYong et al. Chapter 13, in Fluoropolymers 1, Synthesis, Hougham etal., Eds., Kluwer, N.Y., 1999). It is further known that fluoropolymersare swelled or dissolved in supercritical CO₂. Accordingly, in oneembodiment of this invention, the fluorinated polyelectrolytes areapplied by spraying them from solutions or suspensions in supercriticalCO₂. Such application may proceed with simultaneous or sequentialspraying of positive and negative fluorinated polyelectrolytes usingdifferent reservoirs for each polymer. The preferred concentration offluorinated polyelectrolyte is 0.1 weight % to 10 weight %. Optionally,a small volume fraction of organic solvent such as ethanol or methanolmay be added to the supercritical CO₂ to improve the dispersion of saidfluorinated polyelectrolytes.

In a further embodiment of this invention, a suspension of apolyelectrolyte complex comprising at least one negative fluorinatedpolyelectrolyte and at least one positive fluorinated polyelectrolytesare sprayed onto a surface from supercritical CO₂.

The duration in which the polyelectrolyte solution is typically incontact with the surface it is sprayed upon (i.e., the contact time)varies from a couple of seconds to several minutes to achieve a maximum,or steady-state, thickness. The contact duration is selected based onthe desired relationship between throughput (i.e., the rate at whichalternating layers are created) and layer thickness. Specifically,decreasing the contact duration increases throughput and decreases layerthickness whereas increasing the duration decreases throughput andincreases thickness. Preferably, the contact time is selected tomaximize the throughput of layers that have a satisfactory thickness andare uniform across the surface.

Other preferred methods of depositing the polyelectrolyte solutionsand/or polyelectrolyte complex include casting, dip coating, and doctorblading. Particularly preferred methods are dip coating and spraying.

Optionally, rinsing may be employed to remove nonadsorbedpolyelectrolyte between the application of each polyelectrolytesolution. The rinsing liquid comprises an appropriate solvent (e.g.,water or organic solvent such as alcohol). For water-solublepolyelectrolytes the preferred solvent is water. If the solvent iswater, the rinsing liquid may also comprise an organic modifier (e.g.,ethanol, methanol, or propanol). The concentration of organic modifiercan be as high as less than 100 percent by weight of the rinsing liquid,but is preferably less than about 50 percent by weight. The rinsingliquid may also comprise a salt (e.g., sodium chloride) which is solublein the solvent and the organic modifier, if included in the rinsingliquid. The concentration of salt is preferably below about 10 percentby weight of the rinsing liquid. It should be noted that as theconcentration of organic modifier increases the maximum solubilityconcentration of salt decreases. The rinsing liquid, however, should notcomprise a polyelectrolyte. The rinsing step may be accomplished by anyappropriate means (e.g., flushing, dipping, or spraying). For sprayrinsing, the amount of waste is preferably reduced by recycling thepolymer solutions removed from the surface. Optionally, prior todepositing the second through n^(th) layer of sprayed oppositely chargedpolyelectrolyte solution, the surface of the multilayer structure may bedried.

When performing multilayering by dipping, in order to avoidprecipitation through cross-contamination, at least one of the rinsesteps preferably employs a solvent which mixes with the solvents inwhich the polyelectrolytes are dissolved/dispersed.

Particles with diameters ranging from nanometers to millimeters may alsobe coated with polyelectrolyte complex. If the alternate layering methodis used, it is not practical to coat particles individually. Neither isthe spray method practical, unless particles are larger than about 100μm. Instead, batches of particles are alternately immersed in coatingsolutions, with intervening rinse, as detailed by Caruso and Sukhorukov,Chapter 12 in “Multilayer Thin Films”, G. Decher and J. B. Schlenoff,Eds., Wiley-VCH, Weinheim, 2003. See also Donath et al., U.S. Pat. Pub.No. 2003/0219384.

In a preferred embodiment of this invention, the tissue engineeringaspects of this invention are promoted by coating complex objects withthe polyelectrolyte complex films of this invention. Assemblies of cellsare then allowed to grow into functional tissues within this complexobject. In one embodiment of this invention, the complex object is aporous “scaffold,” or structural support. Preferably, the scaffoldprovides a three dimensional cell growth environment, in which cells arein close proximity such that the cells may self-assemble. See S.Levenberg, et al., “Embryonic Stem Cells in Tissue Engineering,” to besubmitted to HANDBOOK OF EMBRYONIC STEM CELLS, Eds. D. Melton, et al.The entire scaffold is on the order of several millimeters in eachdimension. A scaffold may be, for example, about 5 mm, about 8 mm, andeven up to about 12 mm in each dimension. The scaffold is preferablyporous, having pore diameters as small as about 10 um to pores as largeas about 1000 um in diameter, more preferably between about 100 um toabout 600 um. The pore void volume in the scaffold may be about 40% toabout 90% of the volume of the entire scaffold. See Valentini et al.,U.S. Pat. No. 5,939,323. Said scaffold is typically a porous mesh orfoam or collection of bundled fibers. Said scaffold may be biodegradableor nonbiodegradable. The function of the scaffold is to direct thegrowth of cells from surrounding tissue or the growth of cells seededwithin the porous structure of the scaffold. In the cases where atemporary role for the scaffold is desired, biodegradable scaffoldmaterials are preferred. Polymer scaffolds are well known to thoseskilled in the art, as detailed in R. C. Thomson et al., Chapter 21 in“Principles of Tissue Engineering,” 2^(nd) Ed., Eds. R. P. Lanza, R.Langer, J. Vacanti, Academic Press, San Diego, 2000.

Advantageously, a polyelectrolyte complex thin film coating on ascaffold material represents a small fraction of the total material onwhich cells grow. For example, a polyelectrolyte complex thin film mighthave a thickness of 0.01 micrometer, whereas the article or scaffold onwhich said thin film is deposited might have dimensions of hundreds ofmicrometers or even millimeters.

Preferred synthetic polymers for making biodegradable articles,including scaffolds for in vitro and in vivo tissue cultures, known tothose skilled in the art (see A. Atala et. al “Synthetic BiodegradablePolymer Scaffolds,” Birkhauser Publishers, Boston, 1997, for example),include poly(glycolic acid), poly(lactic acid), poly(β-hydroxybutyrate),poly(caprolactone), polyphosphazenes, poly(propylene fumarate),polyarylates, polyethylene glycol, and copolymers made from theseexamples. Preferred biodegradable polymers from biological sourcesinclude collagen, glycosaminoglycans, hyaluronic acid, chitosan,polyhydroxyalkanoates, alginates, and modified polysaccharides, such ascellulose, starch, and chitin.

Scaffold coated entirely with polyelectrolyte complex comprising asurface layer of fluorinated polyelectrolyte is hydrophobic andtherefore difficult to wet. Preferred scaffolds comprising thin films ofpolyelectrolyte complex comprising fluorinated polymer also compriseareas coated with hydrophilic polyelectrolyte complexes, such aspolyelectrolyte complexes comprising polycarboxylic acid, or a copolymerof carboxylic acid repeat unit, and zwitterionic repeat unit. Thepreferred scaffold is made by intermingling individual fibers ofbiodegradable polymer previously modified with either polyelectrolytecomplex thin films comprising fluorinated (hydrophobic) or hydrophilicrepeat units.

Optionally, said polymer scaffold material also comprises inorganicmaterial known to those skilled in the art to impart mechanical strengthand/or rigidity to the scaffold, such as hydroxyapatite and calciumcarbonate. Preferably, said inorganic material comprises hydroxyapatiteparticles, preferably with an aspect ratio of greater than about 5:1.

Optionally, said polymer scaffold further comprises agents known topromote growth, i.e., growth factors, and other cell nutrients.Optionally, the polymer scaffold may comprise immobilized ligands suchas RGD peptides and the adherent domain of fibronectin, to provide ananchor for cells.

A preferred scaffold for preparing layers of cultured cells is a porousfilm of polyelectrolyte complex described by Hiller et al. (see NatureMat. 1, 59 (2002) and U.S. Pat. Pub. No. 2003/0215626). Such a film iscreated by preparing a polyelectrolyte thin film complex comprising weakacid or weak base units, then changing the state of charge of the weakacid or base in said thin film complex, which results in phaseseparations of polymeric constituents and porosity. For the purposes ofthis invention, such a porous material is rendered suitable forpromoting cell growth by coating it with at least one layer offluorinated polyelectrolyte.

In another embodiment of this invention, the complex object whichsupports growth of cells is a template. A preferred template is a tube,or a plurality of tubes. In a preferred mode of application of thisinvention, the external surface of a tube or fiber, preferably ofdiameter in the range about 1 micrometer to about 1 millimeter, iscoated with the polyelectrolyte complex of this invention. Cells arethen allowed to grow on this tube or fiber. Optionally, the tubecomprises biodegradable material so that it partially or fully degradesover time, whether in vitro or in vivo. An alternative preferred mode ofgrowth on tube templates is to coat the internal surface of a tube andto allow cells to grow on the internal surface of said tube. Optionally,both the internal and external surface of a tube is coated with thepolyelectrolyte complexes of this invention. Preferred materials forsaid tubes or fibers are of the type known to those skilled in the art,and include poly(glycolic acid), poly(lactic acid),poly(β-hydroxybutyrate), poly(caprolactone), polyphosphazenes andcopolymers made from these examples, as well as collagen,glycosaminoglycans, hyaluronic acid, chitosan, polyhydroxyalkanoates,and modified polysaccharides, such as cellulose, starch, and chitin.

In yet another embodiment of the present invention, the polyelectrolytecomplex is a coating or layer on a substrate or substratum and may bedeposited according to any appropriate method (see, e.g., supra, as amultilayer or as a pre-formed polyelectrolyte complex). The substratummay be non-porous or porous and may be comprised of many types ofmaterials that are well known in the art such as polymers, metals, andceramics. The surface of polymeric support materials may be positivelycharged by comprising tetraalkyl ammonium groups, negatively charged bycomprising sulfonate groups, or neutral. In another embodiment thesubstratum is porous and comprises a material selected from the groupconsisting of polypropylene, nylon, polytetrafluoroethylene, glass, andalumina (all of which are known to those of skill in the art).Typically, the average size of the pores is between about 100 nm andabout 10 μm and the degree of porosity is between about 0.1 and about60%. “Degree of porosity” refers to the volume % of the material that isoccupied by pores. Advantageously, when the fluorinated polyelectrolytesof the present invention are applied to a porous substrate, thepolyelectrolytes achieve a high degree of penetration into thesubstrate's pores. For example, a first polyelectrolyte solutioncomprising a charged fluorinated polyelectrolyte may be applied to aporous substrate and the solution allowed to penetrate the pores. Asecond polyelectrolyte solution comprising a charged fluorinatedpolyelectrolyte having a charge opposite to that of the firstpolyelectrolyte solution may then be applied to the porous substrate.The oppositely charged fluorinated polyelectrolytes may then form aninterpenetrating network of complexed fluorinated polyelectrolytes whichis insoluble, which is resilient, and which will not migrate onceapplied to the porous substrate.

In another embodiment the polyelectrolyte complex is a free, orisolated, membrane. Typically, an isolated membrane comprising apolyelectrolyte complex is formed by depositing the complex on a supportand then dissolving the support. For example, a cellulose acetatesupport may be dissolved with acetone to remove it from a multilayercomprising charged particles and polymers. See Mamedov et al., Langmuir16, 5530 (2000). This process typically has characteristics that areoften considered to be drawbacks. For example, it may be slow, ittypically requires disposal of organic solvents, it destroys thesubstratum, it may be difficult or impossible to employ on a multilayermembrane which does not contain charged particles, and it may denature,or deactivate, biologically-derived species (e.g., enzymes) incorporatedwithin the membrane.

Alternatively, isolated membranes may be produced by using a releasestratum that has a composition that is different from the remainder ofthe membrane, the release stratum is designed to decompose, dissociate,or become weakly associated under certain conditions (e.g., a change insalt concentration, pH, and/or temperature) thereby freeing the membranefrom a substratum. This approach was set forth in U.S. Prov. App. Ser.No. 60/284,723, PCT App. No. PCT/US02/11917, and U.S. application Ser.No. 10/475,236 which are hereby incorporated by reference in theirentirety for all purposes. These disclose a releasable membranestructure for producing a free membrane comprising a substratum orsupport and a release stratum deposited on the substratum. In thepresent invention, a membrane stratum comprising the polyelectrolytecomplex of this invention, such as fluorinated polyelectrolytes, orpolyelectrolytes comprising zwitterion repeat units, is deposited on therelease stratum. Each release stratum comprises at least twooppositely-charged polyelectrolytes and is preferably a sequence ofalternating oppositely-charged polyelectrolytes applied as layers.Selective decomposition of the oppositely-charged polyelectrolytes ofthe release stratum affords controlled separation of high quality freemembranes. Examples of release stratum polyelectrolytes and dissociationstimuli include: PSS/PDADMA released by a NaCl solution about 3.5 M;PAA/PDADMA released by a NaCl solution about 0.6 M; andPSS/PDADMA-co-PAA released by a solution having a pH 6. Thus, dependingon the desired polyelectrolyte free membrane, the appropriateoppositely-charged polyelectrolytes may be selected to create a releasestratum that decomposes, dissociates, or becomes weakly associated underconditions which do not negatively impact the integrity of the freemembrane. A preferred release stratum is a multilayer of PDADMA-co-PAArandom copolymer with a PAA content of between 20 and 60 mole % (basedon the polymer repeat unit) layered with PSS under conditions ofsolution and rinse pH of less than about 4. The preferred releasestimulus for this stratum is exposure to solution pH above about 5.

In one embodiment of this invention, a free membrane is created with ahydrophobic stratum on one side and a hydrophilic stratum the otherside. Preferably the hydrophobic stratum comprises fluorinatedpolyelectrolyte. Preferably the hydrophilic stratum comprisespolyacrylic acid, and, more preferably, the hydrophilic stratumcomprises a copolymer of acrylic acid and a zwitterion, where thezwitterion preferably comprises between 10 and 80 mole % of saidcopolymers. Preferably the zwitterion repeat unit is3-[2-(acrylamido)-ethyldimethyl ammonio]propane sulfonate, AEDAPS.Preferably, both sides of the membrane bear the same charge, so thatthere will not be a tendency for the membrane to self-associate.

A free membrane bearing such a fluorinated hydrophobic surface on oneside and a hydrophilic surface on the other side is used advantageouslywhere cell adhesion, growth, and proliferation is required on one sideof the membrane but not on the other side. Thus, a preferred applicationof said membrane is as a wound dressing. In a similar preferredapplication, a film comprising synthetic polymers known to those skilledin the art, such as polyvinyl chloride, polyurethane, polysiloxanes, andother elastic polymers is advantageously coated with a thin film ofpolyelectrolyte complex comprising fluorinated polyelectrolyte. Saidcoated synthetic polyelectrolyte film is also preferably applied as awound dressing. The membrane surface on which the thin film ofpolyelectrolyte complex is coated is preferably in contact with thewound area to encourage cell and tissue growth and healing.

In yet another embodiment of this invention, an area on which cells areto adhere and grow is defined by microcontact printing of fluorinatedpolyelectrolyte to a surface. Microcontact printing is a method, knownto those skilled in the art, whereby a patterned stamp is “inked” with amaterial to be transferred to a surface (for a description, see S.Takayama et al, Chapter 18 in “Principles of Tissue Engineering,” 2^(nd)Ed., Eds. R. P. Lanza, R. Langer, J. Vacanti, Academic Press, San Diego,2000). Small molecules and large molecules may be transferredefficiently from the stamp features, faithfully reproducing the stamppattern, which may have dimensions down to the micrometer range. Formicrocontact printing, the fluorinated polyelectrolyte is preferablyapplied to the surface of the stamp with a cotton swab, allowed to dry,and then pressed on the surface to be patterned. The width of the areaon which cells attach and grow is determined by the width of the raisedareas of the stamp features, from which cytophilic polymer is patterned.For oriented cell growth it is preferable to maintain a sufficientlynarrow width of cytophilic material. Preferred line widths for orientedcell growth are on the order of the cell width. Preferred line widthsfor smooth muscle cells are from 5 to 50 micrometers. For dense packingof oriented cell growth, the cytophilic areas on the surface arepreferably a plurality of closely-spaced parallel lines.

A preferred mode of pattern formation for patterned cell growth is toemploy a stamp inked with fluorinated polyelectrolyte and to stamp saidpolyelectrolyte on the surface of a polyelectrolyte thin film,preferably prepared by the polyelectrolyte multilayer technique,comprising at least one surface layer of polyelectrolyte comprisingzwitterion repeat units. This operation produces a pattern of highhydrophobicity contrast. Cells are then permitted to attach and grow onthe areas comprising fluorinated polyelectrolyte on the surface (fromthe stamp) and are substantially excluded from areas comprising thehydrophilic zwitterionic polyelectrolyte on the surface. Preferredcompositions for patterned growth are in accord with preferredcompositions listed above for encouraging or preventing cell adhesionand growth on surfaces. This strategy of contrasting hydrophobic andhydrophilic areas on a surface leads to highly localized and directedgrowth of cells, as shown in the examples.

In another preferred mode of pattern formation, a patterned stamp isinked with fluorinated polyelectrolyte and said fluorinatedpolyelectrolyte is then stamped onto the surface of a thin film ofpolyelectrolyte complex comprising a zwitterionic repeat unit. Thepattern is then immersed into a solution comprising at least oneprotein. The protein adsorbs to the fluorinated areas and is repelledfrom the zwitterionic areas. The patterned protein is then preferablyemployed as a substrate for cell growth. Preferably, proteins known toenhance the adhesion of cells to surfaces, more preferably fibrinogenand/or fibronectin, are patterned in this manner.

The following examples further illustrate the invention. The abovedescribed polyelectrolytes and additives were used for building thinfilms of a variety of compositions on substrates. The thin films,solutions, and additives were modified in various ways as shown in theexamples, and the effects of those modifications on theinhibition/enhancement of cell adhesion, growth, and differentiationwere monitored. While the number of possible combinations is immense,the goal was to make some broad deductions concerning the role of filmcomposition and modification in the adhesion of cells. The examples areillustrative and not meant to be limiting.

For clarity, the following shorthand for multilayers is used: (A/B)_(x)where A is the starting polyelectrolyte contacting the substrate, B isthe terminating polyelectrolyte in contact with subsequent cellsolutions and x is the number of layer pairs. In (A/B)_(x)A, A would bethe terminating polymer. Salt, MY (cation M and anion Y), has animportant role in the buildup process and is represented by (A/B)_(x)@cMY, where c is the molarity of the salt (MY) in the polymer solution.The pH can be included in the nomenclature especially when using pHdependent PEMUs. For example, (PAH/PAA)₂ @0.25 M NaCl @pH 7.4, representtwo pairs of PAH/PAA built at 0.25 M NaCl and a pH of 7.4.

Example 1 Material Used to Build PEMUs and Characterization Methods

Materials.

1,3 propane sultone (PS) and acrylic acid were obtained from Aldrich.2-(acrylamido)-ethyl dimethyl amine (AEDA) was obtained fromMonomer-Polymer & Dajac Inc. Poly(styrenesulfonic acid), PSS (molecularweight, MW 7.3×10⁴, M_(w)/M_(n)=1.06), poly(diallyldimethylammoniumchloride), PDADMAC (MW 3.7×10⁵, M_(w)/M_(n)=2.1), poly(allylaminehydrochloride), PAH (MW ˜7×10⁴) and poly(acrylic acid), PAA (MW˜2.4×10⁵) were used as received from Aldrich. Poly(N-methyl-2-vinylpyridinium iodide-block-ethylene oxide), PM2VP-b-PEO (PM2VP block 86%quarternized, respective block molecular weights 56,500:5900M_(w)/M_(n)=1.08), poly(methacrylic acid-block-ethylene oxide),PMA-b-PEO (respective block molecular weights 41,000:30,700M_(w)/M_(n)=1.5) and poly(4-vinyl pyridine), P4VP (MW ˜5×10⁴) were fromPolymer Source Inc. Nafion® was purchased from Aldrich and used as 2.5wt. % solution in ethanol:methanol 50:50 vol/vol for stamping. Allpolymer, monomer, and buffer solutions were prepared using 18 M Ω water(except fluorinated polymers).

Polyelectrolyte Multilayers on Glass Cover Slips.

Glass cover slips (cover glass, No. 1½, 22 mm sq., Corning) were cleanedin 70% H₂SO₄ (conc.)/30% H₂O_(2(aq)) (“piranha:” caution, piranha is astrong oxidizer and should not be stored in closed containers) then inhot H₂O₂/ammonia/water, 1:1:7 vol/vol, rinsed in water and blown drywith a stream of nitrogen. Polymer solution concentrations were 10 mM(with respect to the monomer repeat unit) in 0.25 M sodium chloride salt(NaCl, Fisher) except for Nafion® which was 0.25 wt. % solution inethanol: methanol 50:50 vol/vol and PFPVP which was 2 mM. Sequentialadsorption of polyelectrolytes on cover slips was performed by handdipping, where the exposure time for the two polymers was 10 minuteswith three rinses of fresh distilled water, 1 minute each, between.

Film Thickness.

The film thickness was determined using a Gaertner Scientific L116Sautogain ellipsometer with 632.8 nm radiation at 70° incident angle. Arefractive index of 1.54 was employed for the multilayer film. Forfluorinated polymers, a refractive index of 1.35 was used. Thicknessesare quoted as “dry” thickness.

Contact Angle Measurements.

Water contact angles measurements were recorded using a contact anglegoniometer (Ramé-Hart, Inc.) with the sessile drop technique.Measurements were done at 5 different locations of the sample andaveraged (RSD 10%). The volume of the drop was maintained at 10 μL.

Polymer Stamping.

Polymer-on-polymer stamping (POPS), as described by Jiang and Hammond(Langmuir, 16, 8501 (2000)), was done with a poly(dimethylsiloxane)stamp, (PDMS), that had defined parallel channels. In this technique,the surface of a patterned PDMS was inked by applying 2.5 wt % Nafion®using a cotton swap, and drying with a nitrogen stream. The patternedside of the stamp was brought in contact with the multilayer surface for10 minutes. The surface was characterized with a KLA-Tencor P15 SurfaceProfiler (KLA-Tencor).

Cell Culture.

The A7r5 rat aortic smooth muscle cells (American Type CultureCollection) were cultured in Dulbecco's modified Eagle medium highglucose supplemented with 10% fetal bovine serum, 100 units mL⁻¹Penicillin G, 100 ug mL⁻¹ Streptomycin and 10 μg mL⁻¹ Gentamicin. Cellswere plated onto 75 mm² culture flasks and maintained at 37° C. in ahumidified atmosphere with 5% CO₂ in air. Cells were fed once per weekand passaged when they reached 85% confluency. Cells were released fromculture flasks using a trypsin and ethylenediamine tetraacetic acid(EDTA) solution in Hanks buffered salt solution (HBSS).

Microscopy.

Equal numbers (˜1×10⁴ cell mL⁻¹) of A7r5 cells were plated onto bare orpolyelectrolyte coated glass cover slips in 6-well dishes and grown for30-48 hours to allow cell attachment before imaging. Live cell phaseimages were obtained using a Zeiss Axiovert-35 microscope equipped witha NEC Ti-24A CCD camera and Metamorph Imaging Software. For staining,cells were washed once with cold phosphate buffered saline pH 7.4 (PBS)and then fixed with ice cold acetone for 1 min. Following 3 washes withPBS, the cells were blocked with 1% bovine serum albumin (BSA) in PBSfor 30 minutes. The cells were stained using 1 unit of Alexa Fluor® 488phalloidin (Molecular Probes, Inc.) in PBS for 20 minutes at roomtemperature. The cells were washed 3 times in PBS and the cover slipswere mounted in Vectashield (Vector Laboratories Inc.) mounting mediumcontaining 1.5 μg mL⁻¹ 4′,6-diamidino-2-phenylindole (DAPI). Stainedcells were observed with a Nikon Microphoto-FX microscope and imagedusing a Zeiss color Axiocam.

Example 2 Polyelectrolyte Synthesis

3-[2-(acrylamido)-ethyl dimethylammonio]propane sulfonate, AEDAPS.

The zwitterionic monomer was made from 2-(acrylamido)-ethyldimethylamine (AEDA) and PS. 200 ppm methyl ethyl hydroquinone inhibitor wasremoved from AA and AEDA by passing the monomer through a column of DTRsilica column (Scientific Polymer Products Inc.). 1.0 equivalent of AEDA(1.42 g, 10 mmol) was dissolved in 28 mL of propylene carbonate (PC).1.1 equivalent of PS (1.34 g, 10.1 mmol) was added to the reactionmixture. The reaction was stirred at 45° C. for 1 hr. The product,3-[2-(acrylamido)-ethyldimethyl ammonio]propane sulfonate, AEDAPS,precipitated and was washed with petroleum ether with a product yield of60%. ¹H NMR (300 MHz, DMSO): δ 8.45 (bs, 1H), 6.15 (m, 2H), 5.55 (m,1H), 3.21 (m, 2H), 3.32-3.59 (m, 6H), 3.06 (s, 6H), 2.47 (t, 2H). FTIR:N—H, 3270 cm⁻¹ (m); C═C—H 3037 cm⁻¹, aliphatic C—H, 2964 cm⁻¹; amideC═O, 1668 cm⁻¹ (s); and 1545 cm⁻¹ (s); C═C stretch 1628 cm⁻¹; S—O, 1200cm⁻¹ (s).

PAA-co-PAEDAPS Copolymer.

The copolymer was made from AEDAPS and AA via free radicalpolymerization. The monomer feed for AA: AEDAPS was 90:10 or 75:25 moleratios. The monomers were dissolved in aqueous 0.5 M NaCl concentrationto assist homogeneity of the copolymer. PAA-co-PAEDAPS (90:10 mol %) wasmade by copolymerizing 2.4 g, 33 mmol AA with 3.2 g, 12 mmol of AEDAPS.Similarly, for PAA-co-PAEDAPS (75:25 mol %), 0.77 g, 11 mmol of AA and4.27 g, 16 mmol were used. Total monomer concentration was 0.9 M in 30mL of distilled water. Potassium persulfate, 7.2 mg (0.1 mol %), wasthen added to the mixture which was heated at 50° C. under argon andstirring for 120 h. The product was dialyzed against distilled waterusing 14,000 molecular-weight-cutoff dialysis tubing. Elemental analysis(E.A., Atlantic Microlab Inc.) for PAA-co-PAEDAPS (90:10 mol %)(C₃H₄O₂)_(0.9)-co-(C₁₀H₂₀N₂O₄S)_(0.1), theory (found): C, 47.98%(46.75%), H 6.14% (6.04%), N, 3.10% (2.30%), S, 3.51% (2.65%). E.A. forPAA-co-PAEDAPS (75:25 mole %), (C₃H₄O₂)_(0.75)-co-(C₁₀H₂₀N₂O₄S)_(0.25),theory (found): C, 47.50% (45.41%), H 6.66% (6.93%), N, 5.83% (5.38%),S, 6.66% (6.60%). The polymers were characterized using FTIR whichconfirmed the presence of both carbonyl stretch C═O (1725 cm⁻¹, AA and1670 cm⁻¹ AEDAPS) and the appearance of sulfonate (ν_(S03-)) stretch at˜1200 cm⁻¹.

P4VTDFOP-co-P4VP Copolymer, “PFPVP”.

1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluoro-8-iodooctane (TDF₁, C₈H₄F₁₃I)was reacted with P4VP to give poly(4-vinyl-trideca-fluoro-octylpyridinium iodide)-co-poly(4-vinyl pyridine). DMF and nitromethane,previously dried using molecular sieves, were used to dissolve thereactants. 1.0 equivalent (1.05 g, 10 mmol) of P4VP was dried at 110° C.for 4 h and then dissolved in a 50 mL 1:1 v/v dry mixture of DMF andnitromethane under stirring conditions for 72 hours at 50° C. 1.2equivalents (5.7 g, 12 mmol) of TDFI was then added to the reactionmixture and left for 48 hours under inert atmosphere. The product wasprecipitated using ethyl acetate then washed with petroleum ether anddried under vacuum for 24 hours at 60° C. E.A for PFPVP,(C₁₅H₁₁NF₁₃I)_(0.45)-co-(C₇H₇N)_(0.55), theory (found): 40.90 (40.95) %C, 3.39 (3.34) % H, 5.06 (5.11) % N, 29.46 (29.54) % F and 17.58 (17.48)% I. The elemental analysis results showed the polymer to be 45±3%quaternized with the fluorinating reagent. Experimental reaction yieldwas 90%. The polymer was characterized by FTIR spectroscopy and wasidentifiable by the distinctive C—F stretch in the 1200 cm⁻¹ region ofthe spectrum.

Example 3 Building up a PEMU Comprising a Zwitterionic Homopolymer

Zwitterionic polyelectrolytes comprise repeat units that bear a negativeand a positive charge. Opposite charges on a repeat unit are inrelatively close proximity and therefore have an opportunity to interactstrongly. Because the zwitterion group is charge balanced (chargeneutral) it does not require counterions when in solution.

Given that opposite charges on zwitterion polymer repeat units interactwith each other, the question arises as to whether polyzwitterions wouldinteract with other charged polymers. If there is no electrostatic orcharge-pairing interaction between molecules, there is no driving forcefor intermolecular attraction and therefore no driving force forpolyelectrolyte complexation, which is required for multilayer buildup.

An attempt was made to construct a multilayer from the zwitterionicpolymer poly(N-propane sulfonate-2-vinyl pyridine), P2PSVP, and anegative or positive polyelectrolyte. For example, P2PSVP and PDADMAwere employed for attempted multilayer buildup at pH 5. Under thiscondition, the multilayer did not build because the negative sulfonateon the P2PSVP interacted with the positive pyridinium on P2PSVP, anintramolecular interaction, rather than with the PDADMA repeat unit(intermolecular interaction). Similarly, multilayers could not beconstructed from P2PSVP and PSS at near-neutral (pH 5-7) conditionsbecause the PSS does not interact sufficiently with the pyridiniumnitrogen on P2PSVP. However, if the pH is lowered below about pH 2,multilayers may be built from PSS and P2PSVP. At this low pH even thestrongly acidic sulfonate groups on P2PSVP are protonated, leaving someof the pyridinium groups unpaired for intermolecular interactions.Multilayers constructed in this way and exposed to higher pH developedporosity and decomposed as a result of the changing internal chargewithin the multilayer.

In another example, PAA, PDADMA, and P2PSVP were employed to make amultilayer. ATR-FTIR was used to check for layer-by-layer buildup.ATR-FTIR monitored the buildup by looking at the characteristics peaksfor the zwitterionic group (sulfonate stretch ν˜1033 cm⁻¹). The firstlayer was PDADMA (Curve A in FIG. 1), which is positively charged. Thesecond layer was the P2PSVP, which was added successfully (Curve B inFIG. 1). The third layer was PAA, which showed the characteristic peaksfor the C═O group in this polymer (Curve C in FIG. 1). The fourth layerwas P2PSVP, which increased the signal for the sulfonate (Curve D inFIG. 1). The fifth layer was PDADMA, but negative peaks appearing in theP2PSVP region (Curve E in FIG. 1) indicated loss of all multilayerzwitterion, showing the P2PSVP was knocked off the surface and replacedby PDADMA. The loss of P2PSVP occurred every time PDADMA was added.Thus, it is clearly shown that polyelectrolyte bearing zwitterionicrepeat units only do not form stable multilayers.

Example 4 Building up a PEMU Comprising a Zwitterionic Repeat UnitCo-Polymerized with a Charged Repeat Unit

By contrast, stable multilayers could be built with a copolymercomprising both zwitterion repeat units and charged repeat units, suchas acrylic acid. FIG. 2 shows the FTIR of the characteristic zwitterionpeaks in poly(acrylic acid)-co-poly(3-[2-(acrylamido)-ethyldimethylammonio]propane sulfonate), PAA-co-PAEDAPS, copolymer growing as anumber of added layers, for a multilayer with the positivepolyelectrolyte PAH. This clearly shows how the copolymer can be used inlayer-by-layer buildup in contrary to the pure zwitterion polymer. FIG.3 shows, by ellipsometry, the layer-by-layer buildup of a PEMU usingPAA-co-PAEDAPS. Thus, it is shown that the net negative charge on thezwitterion-bearing polyelectrolyte copolymer stabilizes the multilayerby providing ion pairing interaction points with oppositely chargedgroups on other polyelectrolyte molecules.

Example 5 Multilayer Thickness, Surface Charge, and Contact Angles

A selection of polyelectrolytes, summarized in Table VI, was employed toprepare various multilayers. Various contact angles were obtained, withthe multilayers comprising a fluorinated polyelectrolyte as the outerlayer having the highest contact angles (most hydrophobic).

TABLE VI Thickness, Surface Charge and Contact Angles ofPolyelectrolytes Surface Contact Multilayer Thickness, Å Charge Angle(PAH/PAA)₂ 125 ± 6  − 5 ± 2° (PAH/PAA)₂PAH 181 ± 13 + 9 ± 2°(PAH/PAA-co-PAEDAPS)₂ 131 ± 3  − 10 ± 2°  (PAH/PAA-co-PAEDAPS)₂PAH 175 ±11 + 12 ± 2°  (P2VMP-b-PEO/PMA-b-PEO)₂  22 ± 4  − 15 ± 2°  (P2VMP-b-PEO/ 37 ± 2  + 20 ± 2°  PMA-b-PEO)₂P2VMP-b-PEO (PDADMA/PSS)₂  38 ± 2  − 30 ±3°  (PDADMA/PSS)₂PDADMA  55 ± 4  + 55 ± 5°  (PFPVP/Nafion)₂ 116 ± 6  −100 ± 5°  (PFPVP/Nafion)₂PFPVP 186 ± 11 + 100 ± 5° 

Example 6 Growth of Smooth Muscle Cells on Polyelectrolyte Multilayers

Polyelectrolyte multilayers were fabricated to test the effect of bothsurface charge and surface hydrophobicity on smooth muscle cellattachment and spreading. Using a panel of negatively and positivelycharged surfaces with varying hydrophobicities, we developed both celladhesive and cell repulsive surfaces. Both types of surfaces may beuseful for different types of biological applications.

The A7r5 cells are vascular smooth muscle cells originally derived fromrat aortic tissue. Smooth muscle cells were chosen considering thepotential application of polyelectrolyte multilayers as coatings forstents, where the problem of restenosis is at least partially caused bythe invasion of vascular smooth muscle cells onto implanted stents. Ahighly cell resistant surface would be of use as a coating forimplantable devices. We found that the nature of the polyelectrolytesurface controls whether the cells become highly spread, non-motile, andcontractile with prominent stress fiber-like actin structures or morerounded and highly motile with actin filament-rich lamellipodia andfilopodia. These different morphology-motility states reflect those ofthe ‘contractile’ and ‘synthetic’ phenotypes of smooth muscle cells.

A sampling of polyelectrolyte surfaces were chosen to explore the effectof charge and the hydrophobicity of the surface on smooth muscle cellmorphology and motility. Table VI (above, Example 5) shows the measuredcontact angle (a measure of hydrophobicity) and thickness of thesurfaces tested.

A7r5 cells cultured on these layers exhibited graded responses withrespect to hydrophobicity on both negatively and positively chargedpolyelectrolyte surfaces, with the negatively charged surfaces beingmore cell-resistant than the positively charged surfaces of comparableor greater hydrophobicity (FIG. 4). A7r5 cells cultured on Nafion® andPFPVP, which differ in their surface charges but are similar inhydrophobicity with contact angles of 100°, exhibit the flat and spreadappearance of the smooth muscle cell ‘contractile’ phenotype. The effectof the charge difference between Nafion® and PFPVP appears to beminimal, as the cell shapes appear nearly identical on both surfaces(FIG. 4A, 4D). Although well spread, the cells cultured on PSS (FIG. 4B)display more edge ‘ruffling’ than those on Nafion®, suggesting that thePSS cells have a more dynamic intracellular organization than theNafion® cells. The PDADMA surface (FIG. 4E) although more hydrophobicthan the oppositely charged PSS (FIG. 4B) appears to be more cellresistant. Many of the cells plated on the PDADMA surface failed toattach at all and those that did appear to be very active with cellularprocesses extending out in all directions. A7r5 cells cultured on themost hydrophilic surfaces, PAA (5±2°, FIG. 4C) and PAH (9±2°, FIG. 4F),appear to be motile, with little spreading and multiple filopodia. PAH,the most hydrophilic positively charged surface tested, appears to bemore cell resistant than PAA. The morphology and motility of these cellsis similar to smooth muscle cells of the ‘synthetic’ phenotype.

Other studies have shown that the rigidity of a substrate can affectcell attachment and alter cell shape. In this investigation, cellmorphology and motility depended more on the hydrophobicity and chargeof the top polyelectrolyte layer than on the thickness of the layers(Table VI). The thickness of each layer reported here was measured whendry. It is known however, that PEMUs swell when wet, and layers maybecome spongy (except for fluorinated polymers which are veryhydrophobic). Comparison of cells grown on layers of similar drythickness (FIGS. 4, A and C, B and C, D and F) demonstrates a distinctlack of cell morphology dependence on surface thickness.

Example 7 Smooth Muscle Cells on Copolymers Comprising Hydrophilic Units

Multilayers of diblock polyelectrolyte polymers and copolymers producedsome of the more pronounced and interesting effects on cell attachment(FIG. 5). PEMUs generated using two diblock polymers, each containingpoly(ethylene oxide), PEO, with one containing PM2VP(poly(N-methyl-2-vinyl pyridinium iodide), FIG. 5A) and the othercontaining PMA (poly(methacrylic acid), FIG. 5B), also produced cellresistant hydrophilic surfaces. The cells cultured on these diblockpolyelectrolytes were highly rounded, loosely attached, and had manyfilopodia. The positively charged diblock polymer (FIG. 5B) appeared tobe more cell resistant than the negatively charged PMA surface (FIG.5A).

Example 8 Cell Resistant Polyelectrolyte Complex Thin Films ComprisingZwitterionic Repeat Units

Polymer surfaces with exposed zwitterion groups may mimic certainbiological surfaces better than uniformly charged surfaces, because thehead groups of three of the four major membrane phospholipids arezwitterions. The effect of a zwitterionic polymer surface on cellattachment was tested using copolymers containing AEDAPS, a zwitterionsynthesized for this purpose, and acrylic acid, AA. As shown above (FIG.4C), cells grown on the experimental control PAA surface were lessspread than the more hydrophobic negatively charged surfaces, but cellsadhered well and many displayed the prominent leading edge filopodiacharacteristic of motile cells (FIG. 6A). Our PAA-terminated(PAH/PAA)_(x) multilayers appear to be more cell adhesive than thosedisclosed by Mendelson et al., (see Biomacromolecules, 4, 96 (2003))possibly due to differences in the deposition conditions (pH), whichmaximized the proportion of PAA in their multilayers. Inclusion ofAEDAPS in the copolymer made the surfaces even more cell resistant. On a90:10 mol % PAA:PAEDAPS copolymer, the cells appeared to be much moreloosely attached and have adopted a spiky appearance associated withactive filopodia (FIG. 6B). Increasing the amount of the zwitterion inthe copolymer to 75:25 mol % PAA:PAEDAPS yielded an extremely cellresistant surface. Indeed, the plated cells failed to attach and insteadclumped together in non-adherent clusters that floated in the culturemedium above the surface.

Example 9 Patterning of a Multilayer Surface

Micropatterning of cultured cells has been used to investigate theeffect of cell shape on various cell functions. Frequently,micropatterning of cells is accomplished using complex microfabricationtechniques often requiring masking of patterned areas. The technique ofpolymer-on-polymer stamping makes the task of micropatterningpolyelectrolyte multilayers a simpler process. For polymer-on-polymerstamping, a polyelectrolyte applied to a patterned stamp can betransferred to a polyelectrolyte multilayer surface of the same oropposite charge. In this investigation, we used a PDMS stamp in which 20μm wide ridges were generated by cutting 80 μm wide grooves. Thesestamps were used to create cell-adhesive 20 μm wide lines of Nafion® ona cell resistant surface of 75:25 mol % PAA:PAEDAPS. When presented withthis micropatterned surface, the A7r5 cells adhered only to the lines ofNafion®. Nuclear staining of cells on these surfaces revealed a regulardistribution of elongate cells along the Nafion® lines (FIG. 7B).Phalloidin staining of the actin filaments in the cells revealed adistinct orientation along the Nafion® stripe, demonstrating that notonly the cell shape can be guided by the stamped surface, but also theunderlying organization of the cell cytoskeleton (FIG. 7A).

Example 10 Modification of Cell Phenotype by Multilayer Surface

The peripheral regions of the PDMS stamp created a non-grooved Nafion®surface for a direct side-by-side comparison of cells growing on twodifferent continuous layers of polyelectrolytes (FIG. 8). Analysis ofcell behavior on these contrasting layers confirmed two importantproperties of the PEMUs. A single layer is sufficient to create adistinct cell phenotype, regardless of the composition of the underlyingsurface. Cells adhered and spread to the same degree on Nafion® stampedas a single layer on underlying surfaces with different properties ofcell resistance —PAA:AEDAPS (FIG. 8A) or (PAH/PAA)₂PAH (FIG. 8B)—or on acontinuous layer of Nafion® overlying PFPVP (FIG. 4A).

This side-by-side analysis of cells on contrasting surfaces alsorevealed that the nature of the PEMU surface has a dramatic affect onthe organization of the cell actin cytoskeleton. The cells associatedwith Nafion® stamped on PAH adopted very different cytoskeletalarrangements (FIG. 8C). Actin filaments in the cells on the PAH surfacewere located primarily in the spiky filopodia that are mediating thecell-surface interaction. This arrangement of actin filaments isconsistent with the cells being primarily the non-muscle motile‘synthetic’ phenotype.

In contrast, the actin filaments in the cells growing on the Nafion®surface are associated primarily with long stress fiber-like structures(FIG. 8C). It is likely that the cells growing on the Nafion® surfaceare in the ‘contractile’ phenotype and the ordered actin bundles are apart of the smooth muscle contractile apparatus. Clearly, thepolyelectrolyte surface is capable of modulating the appearance of notonly the cell adherence and spreading but also the underlyingarrangement of the cellular cytoskeleton.

Example 11 Controlling Platelet Adhesion

Development of more effective antifouling surfaces for blood contactingbioimplantable devices is of critical importance. Of particular interestis finding effective cell repellent surfaces to combat the problem ofrestenosis. Restenosis involves the progressive occlusion of blood flowcaused by the buildup of cells and debris on an implanted stent.Adherence of platelets to implanted stents is one of the earliest stepsleading to restenosis. We have developed and tested polyelectrolytemultilayer surfaces for their ability to resist platelet activation andadherence. Freshly isolated human platelets were washed extensively andincubated in an activated or unactivated state on polyelectrolyte coatedcoverslips for a period of sixty minutes at 37° C. Following theincubation period, the coverslips were gently washed, fixed, and stainedusing Alexa 488 labeled phalloidin to detect filamentous actin presentin activated platelets. The coverslips were then imaged using standardfluorescence microscopy techniques. Platelets pre-activated with 20 mMADP, a standard technique for activating platelets, adhered in similarnumbers and states of spreading on all the tested surfaces, except 25%AEDAPS, to which preactivated platelets adhered poorly. In contrast,when unactivated platelets were incubated with polyelectrolyte-coatedcoverslips, significant differences were detected in the number thatbecame activated and attached. Approximately the same number ofplatelets adhered to Nafion, a negatively charged hydrophobic surfaceand PAH, a positively charged hydrophilic surface, whether the plateletswere pre-activated or not. Fewer pre-activated platelets adhered tosurfaces of PAA and a 90:10 mol % copolymer mixture of PAA:AEDAPS thanto the PAH and Nafion surfaces. Moreover, fewer unactivated thanpre-activated platelets adhered to PAA surface and 90:10 PAA:AEDAPS. Themost dramatic difference was observed for the 75:25 mol % copolymermixture of PAA:AEDAPS, which had only sparsely attached platelets whenincubated with unactivated platelets relative to the other surfaces(FIG. 9E). The PAA, 90:10 mol % PAA:AEDAPS and 75:25 mol % PAA:AEDAPSsurfaces we have developed are resistant to plateletadherence/activation, with the 75:25 mol % PAA:AEDAPS, beingparticularly effective.

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

When introducing elements of the present invention or the preferredembodiment(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

As various changes could be made in the above without departing from thescope of the invention, it is intended that all matter contained in theabove description and shown in the accompanying drawings shall beinterpreted as illustrative and not in a limiting sense.

What is claimed is:
 1. An article adapted for use in combination withliving tissue or organisms, the article comprising a polyelectrolytefilm, the film comprising a bulk region comprising an interpenetratingnetwork of a net positively charged polyelectrolyte polymer and a netnegatively charged polyelectrolyte polymer, the film further comprisinga first surface region and a second surface region with a net positivelycharged polyelectrolyte polymer or net negatively chargedpolyelectrolyte polymer exposed at each of said first surface region andsecond surface region wherein (1) the net positively chargedpolyelectrolyte polymer or the net negatively charged polyelectrolytepolymer exposed in said first surface region contains a polymer repeatunit having at least two fluorine atoms and (2) the net positivelycharged polyelectrolyte polymer or the net negatively chargedpolyelectrolyte polymer exposed in said second surface region contains apolymer repeat unit having a zwitterion group and a charged polymerrepeat unit that is non-zwitterionic.
 2. The article of claim 1 whereinthe article comprises the polyelectrolyte film and a metal substratumwith the first surface region contacting the metal substratum.
 3. Thearticle of claim 1 wherein the article is a membrane having opposingsides, the first surface region being one of the opposing sides and thesecond surface region being the other opposing side.
 4. The article ofclaim 3 further comprising a film comprising a synthetic elastomericpolymer selected from the group consisting of polyvinyl chloride,polyurethane, polysiloxanes, and combinations thereof said filmcomprising said synthetic elastomeric polymer in contact with said firstsurface region.
 5. The article of claim 1 wherein the first surfaceregion and the second surface region have substantially planar exposedsurfaces wherein the first and second surface regions are substantiallycontiguous so as to define a pattern of regions having water contactangles that differ by at least 30 degrees.
 6. The article of claim 5wherein the first surface region has a dimensional aspect between 1micrometer and 1000 micrometers.
 7. The article of claim 5 wherein thefirst surface region is stamped onto the second surface region.
 8. Amethod for controlling the attachment and growth of cells on a surfaceof an article, the method comprising contacting the article with livingtissue, living organisms, or with water in an aqueous system comprisingliving organisms wherein the article comprises a substratum having asurface and a film on the surface, the film comprising a bulk regioncomprising an interpenetrating network of a net positively chargedpolyelectrolyte polymer and a net negatively charged polyelectrolytepolymer, and wherein the film comprises a first surface region and asecond surface region with a net positively charged polyelectrolytepolymer or net negatively charged polyelectrolyte polymer exposed ateach of said first surface region and second surface region, wherein (1)the net positively charged polyelectrolyte polymer or net negativelycharged polyelectrolyte polymer exposed in said first surface regioncontains a polymer repeat unit having at least two fluorine atoms and(2) the net positively charged polyelectrolyte polymer or net negativelycharged polyelectrolyte polymer exposed in said second surface regioncontains a polymer repeat unit having a zwitterion group and a chargedpolymer repeat unit that is non-zwitterionic and wherein the netpositively charged polyelectrolyte polymer or net negatively chargedpolyelectrolyte polymer exposed in said second surface region is acopolymer comprising between about 20 mole % and about 70 mole % ofpolymer repeat units having zwitterion groups.
 9. The method of claim 8wherein the first surface region of the film promotes the attachment andgrowth of cells and the second surface region inhibits the attachmentand growth of cells.
 10. The article of claim 1 wherein thepolyelectrolyte film is in contact with a substratum selected from thegroup consisting of stents, catheters, vascular grafts, vascularprostheses, contact lenses, intraocular implants, artificial valves forin vivo use, artificial organs, dental implants, metal implants intobone, corneal implants, Petri dishes, roller bottles, microcarriers,porous structural supports for three dimensional cell growth, syntheticelastomeric polymers, and metal objects adapted for use in aqueoussystems containing living organisms.
 11. The article of claim 1 whereinthe zwitterion group is selected from the group consisting of:N,N-dimethyl-N-acryloyloxyethyl-N-(3-sulfopropyl)-ammonium betaine,N,N-dimethyl-N-acrylamidopropyl-N-(2-carboxymethyl)-ammonium betaine,N,N-dimethyl-N-acrylamidopropyl-N-(3-sulfopropyl)-ammonium betaine,N,N-dimethyl-N-acrylamidopropyl-N-(2-carboxymethyl)-ammonium betaine,2-(methylthio)ethyl methacryloyl-S-(sulfopropyl)-sulfonium betaine,2-[(2-acryloylethyl)dimethylammonio]ethyl 2-methyl phosphate,2-(acryloyloxyethyl)-2′-(trimethylammonium)ethyl phosphate,[(2-acryloylethyl)dimethylammonio]methyl phosphonic acid,2-methacryloyloxyethyl phosphorylcholine (MPC),2-[(3-acrylamidopropyl)dimethylammonio]ethyl 2′-isopropyl phosphate,1-vinyl-3-(3-sulfopropyl)imidazolium hydroxide,(2-acryloxyethyl)carboxymethyl methylsulfonium chloride,1-(3-sulfopropyl)-2-vinylpyridinium betaine,N-(4-sulfobutyl)-N-methyl-N,N-diallylamine ammonium betaine,N,N-diallyl-N-methyl-N-(2-sulfoethyl)ammonium betaine,N,N-dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl)ammoniumbetaine, N,N-dimethyl-N-acryloyloxyethyl-N-(3-sulfopropyl)-ammoniumbetaine, N,N-dimethyl-N-acrylamidopropyl-N-(2-carboxymethyl)-ammoniumbetaine, N,N-dimethyl-N-methacryloyloxyethyl-N-(3-sulfopropyl)-ammoniumbetaine,N,N-dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl)ammoniumbetaine, and copolymers thereof.
 12. The article of claim 1 wherein thenet positively charged polyelectrolyte polymer or the net negativelycharged polyelectrolyte polymer exposed in said second surface regioncontaining the polymer repeat unit having the zwitterion group furthercomprises between about 10% and about 90% the non-zwitterionic chargedpolymer repeat unit.
 13. An article adapted for use in combination withliving tissue or organisms, the article comprising a polyelectrolytefilm, the film comprising a bulk region comprising an interpenetratingnetwork of a net positively charged polyelectrolyte polymer and a netnegatively charged polyelectrolyte polymer, the film further comprisinga first surface region and a second surface region with a net positivelycharged polyelectrolyte polymer or net negatively chargedpolyelectrolyte polymer exposed at each of said first surface region andsecond surface region for forming an interface with a cell growth mediumat each of said first and second surface regions, wherein (1) the netpositively charged polyelectrolyte polymer or the net negatively chargedpolyelectrolyte polymer exposed in said first surface region contains apolymer repeat unit having at least two fluorine atoms and (2) the netpositively charged polyelectrolyte polymer or the net negatively chargedpolyelectrolyte polymer exposed in said second surface region contains apolymer repeat unit having a zwitterion group and a non-zwitterioniccharged polymer repeat unit.
 14. An article as set forth in claim 13wherein said polyelectrolyte film is stable in an aqueous environmenthaving a pH of 7.4.
 15. An article adapted for use in combination withliving tissue or organisms, the article comprising a polyelectrolytefilm that is stable in an aqueous environment having a pH of 7.4, thefilm comprising a bulk region comprising an interpenetrating network ofa net positively charged polyelectrolyte polymer and a net negativelycharged polyelectrolyte polymer, the film further comprising a firstsurface region and a second surface region with a net positively chargedpolyelectrolyte polymer or net negatively charged polyelectrolytepolymer exposed at each of said first surface region and second surfaceregion wherein (1) the net positively charged polyelectrolyte polymer orthe net negatively charged polyelectrolyte polymer exposed in said firstsurface region contains a polymer repeat unit having at least twofluorine atoms and (2) the net positively charged polyelectrolytepolymer or the net negatively charged polyelectrolyte polymer exposed insaid second surface region contains a polymer repeat unit having azwitterion group and a non-zwitterionic charged polymer repeat unit. 16.The method of claim 8 wherein the substratum is selected from the groupconsisting of stents, catheters, vascular grafts, vascular prostheses,contact lenses, intraocular implants, artificial valves for in vivo use,artificial organs, dental implants, metal implants into bone, cornealimplants, Petri dishes, roller bottles, microcarriers, porous structuralsupports for three dimensional cell growth, synthetic elastomericpolymers, and metal objects adapted for use in aqueous systemscontaining living organisms.
 17. The method of claim 8 wherein thearticle comprises the polyelectrolyte film and a metal substratum withthe first surface region contacting the metal substratum.
 18. The methodof claim 8 wherein the article is a membrane having opposing sides, thefirst surface region being one of the opposing sides and the secondsurface region being the other opposing side.
 19. The method of claim 8wherein the article further comprises a film comprising a syntheticelastomeric polymer selected from the group consisting of polyvinylchloride, polyurethane, polysiloxanes, and combinations thereof, saidfilm comprising said synthetic elastomeric polymer in contact with saidfirst surface region.
 20. The method of claim 8 wherein the firstsurface region and the second surface region have substantially planarexposed surfaces wherein the first and second surface regions aresubstantially contiguous so as to define a pattern of regions havingwater contact angles that differ by at least 30 degrees.